U.S. patent number 10,933,129 [Application Number 13/560,955] was granted by the patent office on 2021-03-02 for methods for administering synthetic nanocarriers that generate humoral and cytotoxic t lymphocyte responses.
This patent grant is currently assigned to Selecta Biosciences, Inc.. The grantee listed for this patent is David H. Altreuter, Petr Ilyinskii, Conlin O'Neil. Invention is credited to David H. Altreuter, Petr Ilyinskii, Conlin O'Neil.
United States Patent |
10,933,129 |
Altreuter , et al. |
March 2, 2021 |
Methods for administering synthetic nanocarriers that generate
humoral and cytotoxic T lymphocyte responses
Abstract
Disclosed are methods for generating humoral and cytotoxic T
lymphocyte (CTL) immune responses in a subject and related
compositions.
Inventors: |
Altreuter; David H. (Wayland,
MA), O'Neil; Conlin (Andover, MA), Ilyinskii; Petr
(Cambridge, MA) |
Applicant: |
Name |
City |
State |
Country |
Type |
Altreuter; David H.
O'Neil; Conlin
Ilyinskii; Petr |
Wayland
Andover
Cambridge |
MA
MA
MA |
US
US
US |
|
|
Assignee: |
Selecta Biosciences, Inc.
(Watertown, MA)
|
Family
ID: |
1000005392015 |
Appl.
No.: |
13/560,955 |
Filed: |
July 27, 2012 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20130028941 A1 |
Jan 31, 2013 |
|
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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61513496 |
Jul 29, 2011 |
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61513526 |
Jul 29, 2011 |
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61513527 |
Jul 29, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K
47/645 (20170801); A61K 39/385 (20130101); A61K
39/39 (20130101); A61K 47/59 (20170801); A61K
47/593 (20170801); A61K 2039/55555 (20130101); Y02A
50/30 (20180101); Y10T 428/2982 (20150115) |
Current International
Class: |
A61K
39/395 (20060101); A61K 47/64 (20170101); A61K
39/39 (20060101); A61K 39/385 (20060101); A61K
47/59 (20170101); A61K 39/00 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
1377279 |
|
Oct 2002 |
|
CN |
|
1692943 |
|
Nov 2005 |
|
CN |
|
1 221 955 |
|
Nov 2005 |
|
EP |
|
1 752 141 |
|
Feb 2007 |
|
EP |
|
WO 95/22963 |
|
Aug 1995 |
|
WO |
|
WO 96/20698 |
|
Jul 1996 |
|
WO |
|
WO 97/04747 |
|
Feb 1997 |
|
WO |
|
WO 97/41440 |
|
Nov 1997 |
|
WO |
|
WO 98/37919 |
|
Sep 1998 |
|
WO |
|
WO 98/40100 |
|
Sep 1998 |
|
WO |
|
WO 98/52581 |
|
Nov 1998 |
|
WO |
|
WO 99/56755 |
|
Nov 1999 |
|
WO |
|
WO 00/06123 |
|
Feb 2000 |
|
WO |
|
WO 00/27363 |
|
May 2000 |
|
WO |
|
WO 00/32227 |
|
Jun 2000 |
|
WO |
|
WO 00/32626 |
|
Jun 2000 |
|
WO |
|
WO 00/50075 |
|
Aug 2000 |
|
WO |
|
WO 01/68103 |
|
Sep 2001 |
|
WO |
|
WO 01/85208 |
|
Nov 2001 |
|
WO |
|
WO 02/32450 |
|
Apr 2002 |
|
WO |
|
WO 02/56905 |
|
Jul 2002 |
|
WO |
|
WO 02/56907 |
|
Jul 2002 |
|
WO |
|
WO 2003/039225 |
|
May 2003 |
|
WO |
|
WO 2003/040164 |
|
May 2003 |
|
WO |
|
WO 2003/086280 |
|
Oct 2003 |
|
WO |
|
WO 2004/007538 |
|
Jan 2004 |
|
WO |
|
WO 2004/016282 |
|
Feb 2004 |
|
WO |
|
WO 2004/022594 |
|
Mar 2004 |
|
WO |
|
WO 2004/030608 |
|
Apr 2004 |
|
WO |
|
WO 2004/053104 |
|
Jun 2004 |
|
WO |
|
WO 2004/058179 |
|
Jul 2004 |
|
WO |
|
WO 2004/071493 |
|
Aug 2004 |
|
WO |
|
WO 2004/084871 |
|
Oct 2004 |
|
WO |
|
WO 2004/098509 |
|
Nov 2004 |
|
WO |
|
WO 2005/014110 |
|
Feb 2005 |
|
WO |
|
WO 2005/042018 |
|
May 2005 |
|
WO |
|
WO 2005/097993 |
|
Oct 2005 |
|
WO |
|
WO 2005/108425 |
|
Nov 2005 |
|
WO |
|
WO 2005/110013 |
|
Nov 2005 |
|
WO |
|
WO 2005/120574 |
|
Dec 2005 |
|
WO |
|
WO 2006/031878 |
|
Mar 2006 |
|
WO |
|
WO 2006/037979 |
|
Apr 2006 |
|
WO |
|
WO 2006/045796 |
|
May 2006 |
|
WO |
|
WO 2006/045849 |
|
May 2006 |
|
WO |
|
WO 2006/063974 |
|
Jun 2006 |
|
WO |
|
WO 2006/066158 |
|
Jun 2006 |
|
WO |
|
WO 2006/102395 |
|
Sep 2006 |
|
WO |
|
WO 2006/117217 |
|
Nov 2006 |
|
WO |
|
WO 2006/135434 |
|
Dec 2006 |
|
WO |
|
WO 2006/137934 |
|
Dec 2006 |
|
WO |
|
WO 2007/001448 |
|
Jan 2007 |
|
WO |
|
WO 2007/003054 |
|
Jan 2007 |
|
WO |
|
WO 2007/019678 |
|
Feb 2007 |
|
WO |
|
WO 2007/062107 |
|
May 2007 |
|
WO |
|
WO 2007/068747 |
|
Jun 2007 |
|
WO |
|
WO 2007/070682 |
|
Jun 2007 |
|
WO |
|
WO 2007/089870 |
|
Aug 2007 |
|
WO |
|
WO 2007/098254 |
|
Aug 2007 |
|
WO |
|
WO 2007/109810 |
|
Sep 2007 |
|
WO |
|
WO 2007/118653 |
|
Oct 2007 |
|
WO |
|
WO 2007/133807 |
|
Nov 2007 |
|
WO |
|
WO 2007/137117 |
|
Nov 2007 |
|
WO |
|
WO 2007/144150 |
|
Dec 2007 |
|
WO |
|
WO 2007/149802 |
|
Dec 2007 |
|
WO |
|
WO 2007/150030 |
|
Dec 2007 |
|
WO |
|
WO 2008/033432 |
|
Mar 2008 |
|
WO |
|
WO 2008/051245 |
|
May 2008 |
|
WO |
|
WO 2008/071774 |
|
Jun 2008 |
|
WO |
|
WO 2008/079924 |
|
Jul 2008 |
|
WO |
|
WO 2008/093173 |
|
Aug 2008 |
|
WO |
|
WO 2008/105773 |
|
Sep 2008 |
|
WO |
|
WO 2008/115319 |
|
Sep 2008 |
|
WO |
|
WO 2008/115641 |
|
Sep 2008 |
|
WO |
|
WO 2008/019142 |
|
Oct 2008 |
|
WO |
|
WO 2008/118861 |
|
Oct 2008 |
|
WO |
|
WO 2008/121926 |
|
Oct 2008 |
|
WO |
|
WO 2008/124632 |
|
Oct 2008 |
|
WO |
|
WO 2008/124634 |
|
Oct 2008 |
|
WO |
|
WO 2008/124639 |
|
Oct 2008 |
|
WO |
|
WO 2008/127532 |
|
Oct 2008 |
|
WO |
|
WO 2008/129020 |
|
Oct 2008 |
|
WO |
|
WO 2008/143709 |
|
Nov 2008 |
|
WO |
|
WO 2008/147456 |
|
Dec 2008 |
|
WO |
|
WO 2009/022154 |
|
Feb 2009 |
|
WO |
|
WO 2009/051837 |
|
Apr 2009 |
|
WO |
|
WO 2009/076158 |
|
Jun 2009 |
|
WO |
|
WO 2009/106999 |
|
Sep 2009 |
|
WO |
|
WO 2009/108822 |
|
Sep 2009 |
|
WO |
|
WO 2009/109428 |
|
Sep 2009 |
|
WO |
|
WO 2009/111588 |
|
Sep 2009 |
|
WO |
|
WO 2008/157419 |
|
Dec 2009 |
|
WO |
|
WO 2010/003009 |
|
Jan 2010 |
|
WO |
|
WO 2010/017330 |
|
Feb 2010 |
|
WO |
|
WO 2010/018130 |
|
Feb 2010 |
|
WO |
|
WO 2010/018131 |
|
Feb 2010 |
|
WO |
|
WO 2010/018132 |
|
Feb 2010 |
|
WO |
|
WO 2010/018133 |
|
Feb 2010 |
|
WO |
|
WO 2010/018384 |
|
Feb 2010 |
|
WO |
|
WO 2010/025324 |
|
Mar 2010 |
|
WO |
|
WO 2010/037402 |
|
Apr 2010 |
|
WO |
|
WO 2010/042863 |
|
Apr 2010 |
|
WO |
|
WO 2010/042866 |
|
Apr 2010 |
|
WO |
|
WO 2010/042870 |
|
Apr 2010 |
|
WO |
|
WO 2010/042876 |
|
Apr 2010 |
|
WO |
|
WO 2010/115046 |
|
Oct 2010 |
|
WO |
|
WO 2010/123569 |
|
Oct 2010 |
|
WO |
|
WO 2010/138192 |
|
Dec 2010 |
|
WO |
|
WO 2010/138193 |
|
Dec 2010 |
|
WO |
|
WO 2010/138194 |
|
Dec 2010 |
|
WO |
|
WO 2011/005850 |
|
Jan 2011 |
|
WO |
|
Other References
International Search Report and Written Opinion for
PCT/US2012/048670 dated Feb. 27, 2013. cited by applicant .
International Preliminary Report on Patentability for
PCT/US2012/048670 dated Feb. 13, 2014. cited by applicant .
[No Author Listed] Nanoparticles as Drug Carriers. Ed, Vladimir
Torchilin. Imperial College Press. 2006. 754 pages. cited by
applicant .
Ackerman et al., Cellular mechanisms governing cross-presentation
of exogenous antigens. Nat Immunol. 2004;5(7):678-84. cited by
applicant .
Aime et al., Lanthanide(III) chelates for NMR biomedical
applications. Chemical Society Reviews. 1998;27:19-29. cited by
applicant .
Akaishi et al., Targeting chemotherapy using antibody-combined
liposome against human pancreatic cancer cell-line. The Tohoku
Journal of Experimental Medicine. 1994;175(1):29-42. cited by
applicant .
Alexander et al., Universal influenza DNA vaccine encoding
conserved CD4+ T cell epitopes protects against lethal viral
challenge in HLA-DR transgenic mice. Vaccine. Jan. 8,
2010;28(3):664-72. Epub Nov. 4, 2009. cited by applicant .
Alexis et al., Factors affecting the clearance and biodistribution
of polymeric nanoparticles. Mol Pharm. Jul.-Aug. 2008;5(4):505-15.
Epub Aug. 4, 2008. cited by applicant .
Allen et al., Nano-engineering block copolymer aggregates for drug
delivery. Colloids Surfaces B-Biointerfaces. 1999;16:3-27. cited by
applicant .
Allison, The mode of action of immunological adjuvants. Dev Biol
Stand. 1998;92:3-11. cited by applicant .
Anderson et al., Delivery Systems for Immunomodulatory Proteins and
Peptides. BioDrugs. Jan. 1997;7(1):51-65. cited by applicant .
Anikeeva et al., Quantum dot/peptide-MHC biosensors reveal strong
CD8-dependent cooperation between self and viral antigens that
augment the T cell response. Proc Natl Acad Sci U S A. Nov. 7,
2006;103(45):16846-51. Epub Oct. 31, 2006. cited by applicant .
Asano et al., Targeting activated lymphocytes with lipid
microsphere containing a cytotoxic agent; efficacy of
immunosuppression with a new drug delivery system. J Urology.
2001;165(5)384. Abstact 1571. cited by applicant .
Astete et al., Synthesis and characterization of PLGA
nanoparticles. J Biomat Sci. 2006;17:247-89. cited by applicant
.
Ataman-Onal et al., Surfactant-free anionic PLA nanoparticles
coated with HIV-1 p24 protein induced enhanced cellular and humoral
immune responses in various animal models. J Control Release. May
15, 2006;112(2):175-85. Epub Mar. 6, 2006. cited by applicant .
Avgoustakis, Pegylated poly(lactide) and poly(lactide-co-glycolide)
nanoparticles: preparation, properties and possible applications in
drug delivery. Curr Drug Deliv. Oct. 2004;1(4):321-33. cited by
applicant .
Bachmann et al., T helper cell-independent neutralizing B cell
response against vesicular stomatitis virus: role of antigen
patterns in B cell induction?. Eur J Immunol. 1995;25(12):3445-51.
cited by applicant .
Badiee et al., Coencapsulation of CpG oligodeoxynucleotides with
recombinant Leishmania major stress-inducible protein 1 in liposome
enhances immune response and protection against leishmaniasis in
immunized BALB/c mice. Clin Vaccine Immunol. Apr.
2008;15(4):668-74. Epub Jan. 30, 2008. cited by applicant .
Bae et al., Mixed polymeric micelles for combination cancer
chemotherapy through the concurrent delivery of multiple
chemotherapeutic agents. J Control Release. Oct. 8,
2007;122(3):324-30. Epub Jun. 13, 2007. cited by applicant .
Bagalkot et al., An Aptamer-Doxorubicin Physical Conjugate as a
Novel Targeted Drug-Delivery Platform. Angew Chem Int.
2006;45(48):8149-52. cited by applicant .
Bala et al., PLGA nanoparticles in drug delivery: the state of the
art. Crit Rev Ther Drug Carrier Syst. 2004;21(5):387-422. cited by
applicant .
Barchet et al., Virus-induced interferon alpha production by a
dendritic cell subset in the absence of feedback signaling in vivo.
J Exp Med. 2002;195(4):507-16. cited by applicant .
Barichello et al., Encapsulation of hydrophilic and lipophilic
drugs in PLGA nanoparticles by the nanoprecipitation method. Drug
Dev Ind Pharm. Apr. 1999;25(4):471-6. cited by applicant .
Barve et al., Induction of immune responses and clinical efficacy
in a phase II trial of IDM-2101, a 10-epitope cytotoxic
T-lymphocyte vaccine, in metastatic non-small-cell lung cancer. J
Clin Oncol. Sep. 20, 2008;26(27):4418-25. cited by applicant .
Batista et al., The who, how and where of antigen presentation to B
cells. Nat Rev Immunol. Jan. 2009;9(1):15-27. cited by applicant
.
Bauer et al., Human TLR9 confers responsiveness to bacterial DNA
via species-specific CpG motif recognition. Proc Natl Acad Sci U S
A. Jul. 31, 2001;98(16):9237-42. Epub Jul. 24, 2001. cited by
applicant .
Bayard et al., Hepatitis B virus (HBV)-derived DRB1*0101-restricted
CD4 T-cell epitopes help in the development of HBV-specific CD8+ T
cells in vivo. Vaccine. May 14, 2010;28(22):3818-26. Epub Mar. 31,
2010. cited by applicant .
Beaurepaire et al., Functionalized Fluorescent Oxide Nanoparticles:
Artificial Toxins for Sodium Channel Targeting and Imaging at the
Single-Molecule Level. Nano Letters. 2004;4(11):2079-83. cited by
applicant .
Bei et al., "TAA polyepitope DNA-based vaccines: A potential tool
for cancer therapy." J Biomed Biotech. 2010; 102758:1-12. cited by
applicant .
Bharali, Micro-and Nanoparticles-Based Vaccines for Hepatitis B.
Immune-Mediated Diseases. 2007:415-21. cited by applicant .
Blanco-Prieto et al., Slow delivery of the selective
cholecystokinin agonist pBC 264 into the rat nucleus accumbens
using microspheres. J Neurochem. Dec. 1996;67(6):2417-24. cited by
applicant .
Blander, Phagocytosis and antigen presentation: a partnership
initiated by Toll-like receptors. Ann Rheum Dis. Dec. 2008;67 Suppl
3:iii44-9. cited by applicant .
Boden et al., Regulatory T cells in inflammatory bowel disease.
Curr Opin Gastroenterol. Nov. 2008;24(6):733-41. cited by applicant
.
Boes et al., T-cell engagement of dendritic cells rapidly
rearranges MHC class II transport. Nature. 418(6901):983-988
(2002). cited by applicant .
Bonifaz et al., Efficient targeting of protein antigen to the
dendritic cell receptor DEC-205 in the steady state leads to
antigen presentation on major histocompatibility complex class I
products and peripheral CD8+ T cell tolerance. J Exp Med.
2002;196(12):1627-38. cited by applicant .
Borges et al., Evaluation of the immune response following a short
oral vaccination schedule with hepatitis B antigen encapsulated
into alginate-coated chitosan nanoparticles. Eur J Pharm Sci. Dec.
2007;32(4-5):278-90. Epub Aug. 15, 2007. cited by applicant .
Bourquin et al., Targeting CpG oligonucleotides to the lymph node
by nanoparticles elicits efficient antitumoral immunity. J Immunol.
Sep. 1, 2008;181(5):2990-8. cited by applicant .
Boussif et al., A versatile vector for gene and oligonucleotide
transfer into cells in culture and in vivo: polyethylenimine. Proc
Natl Acad Sci. USA. 1995;92:7297-301. cited by applicant .
Bozzacco et al., DEC-205 receptor on dendritic cells mediates
presentation of HIV gag protein to CD8+ T cells in a spectrum of
human MHC I haplotypes. Proc Natl Acad Sci USA.
2007;104(4):1289-94. cited by applicant .
Brito et al., Nanoparticulate carriers for the treatment of
coronary restenosis. Int J Nanomedicine. 2007;2(2):143-61. cited by
applicant .
Bullis, Shape Matters for Nanoparticles. Technology Review. Aug. 7,
2008. 2 pages. cited by applicant .
Bundy et al., Escherichia coli-based cell-free synthesis of
virus-like particles. Biotechnol Bioeng. May 1, 2008;100(1):28-37.
cited by applicant .
Busson et al., Prediction of CD4(+) T cell epitopes restricted to
HLA-DP4 molecules. J Immunol Methods. Dec. 20,
2006;317(1-2):144-51. Epub Oct. 26, 2006. cited by applicant .
Cameron et al., Aliphatic polyester polymer stars: synthesis,
properties and applications in biomedicine and nanotechnology. Chem
Soc Rev. Mar. 2011;40(3):1761-76. cited by applicant .
Carino et al., Nanosphere based oral insulin delivery. J Control
Release. 2000;65(1-2):261-9. cited by applicant .
Carrasco et al., B cells acquire particulate antigen in a
macrophage-rich area at the boundary between the follicle and the
subcapsular sinus of the lymph node.Immunity. Jul.
2007;27(1):160-71. Epub Jul. 19, 2007. cited by applicant .
Castelli et al., HLA-DP4, the most frequent HLA II molecule,
defines a new supertype of peptide-binding specificity. J Immunol.
Dec. 15, 2002;169(12):6928-34. cited by applicant .
Cerritelli et al., PEG-SS-PPS: reduction-sensitive disulfide block
copolymer vesicles for intracellular drug delivery.
Biomacromolecules. Jun. 2007;8(6):1966-72. Epub May 12, 2007. cited
by applicant .
Chacon et al., Optimized preparation of poly D,L (lactic-glycolic)
microspheres and nanoparticles for oral administration. Intl J
Pharmaceutics. 1996;141:81-91. cited by applicant .
Chapoval et al., HLA-DQ6 and HLA-DQ8 transgenic mice respond to
ragweed allergens and recognize a distinct set of epitopes on short
and giant ragweed group 5 antigens. J Immunol. Aug. 15,
1998;161(4):2032-7. cited by applicant .
Cheng et al., Formulation of functionalized PLGA-PEG nanoparticles
for in vivo targeted drug delivery. Biomaterials.
2007;28(5):869-76. cited by applicant .
Chengalvala et al., Enhanced immunogenicity of hepatitis B surface
antigen by insertion of a helper T cell epitope from tetanus
toxoid. Vaccine. Mar. 5, 1999;17(9-10):1035-41. cited by applicant
.
Chinen et al., Basic and clinical immunology. J Allergy Clin
Immunol. Aug. 2005;116(2):411-8. cited by applicant .
Chu et al., Aptamer mediated siRNA delivery. Nuc Acid Res.
2006;34:e73. cited by applicant .
Chu et al., CpG oligodeoxynucleotides act as adjuvants that switch
on T helper 1 (Th1) immunity. J Exp Med. Nov. 17,
1997;186(10):1623-31. cited by applicant .
Chu et al., Labeling tumor cells with fluorescent
nanocrystal-aptamer bioconjugates. Biosens Bioelectron.
2006;21:1859-66. cited by applicant .
Chukwu et al., Loading some psychopharmacologic agents onto
poly(butylcynoacrylate) nanoparticles--a means for targeting agents
to the brain and improving therapeutic efficiency. Proc Int'l Symp
Control Rd Bioact Mat. 1999:1148-9. cited by applicant .
Clark, The reticulum of lymph nodes in mice studied with the
electron microscope. Am J Anat. 1962;110:217-57. cited by applicant
.
Connor et al., Ex vivo evaluation of anti-EpCAM immunocytokine
huKS-1L2 in ovarian cancer. J Immunother. 2004;27(3):211-19. cited
by applicant .
Conti et al., Thymopentin loaded microsphere preparation by w/o/w
emulsion technique: in vitro/ex vivo evaluation. J Microencapsul.
May-Jun. 1997;14(3):303-10. cited by applicant .
Croy et al., Polymeric micelles for drug delivery. Curr Pharm
Design. 2006;12:4669-84. cited by applicant .
Cruz et al., The influence of PEG chain length and targeting moiety
on antibody-mediated delivery of nanoparticle vaccines to human
dendritic cells. Biomaterials. Oct. 2011;32(28):6791-803. Epub Jul.
2, 2011. E-pub version. cited by applicant .
Cvetanovich et al., Human regulatory T cells in autoimmune
diseases. Curr Opin Immunol. Dec. 2010;22(6):753-60. Epub Sep. 24,
2010. cited by applicant .
Czarniecki, Small molecule modulators of toll-like receptors. J Med
Chem. Nov. 13, 2008;51(21):6621-6. doi: 10.1021/jm800957k. Epub
Oct. 2, 2008. cited by applicant .
Dakappagari et al., A chimeric multi-human epidermal growth factor
receptor-2 B cell epitope peptide vaccine mediates superior
antitumor responses. J Immunol. Apr. 15, 2003;170(8):4242-53. cited
by applicant .
Davis et al., CpG DNA is a potent enhancer of specific immunity in
mice immunized with recombinant hepatitis B surface antigen. J
Immunol. Jan. 15, 1998;160(2):870-6. cited by applicant .
De Gregorio et al., Alum adjuvanticity: unraveling a century old
mystery. Eur J Immunol. Aug. 2008;38(8):2068-71. cited by applicant
.
De Jaeghere et al., Freeze-drying and lyopreservation of diblock
and triblock poly(lactic acid)-poly(ethylene oxide) (PLA-PEO)
copolymer nanoparticles. Pharm Dev Technol. 2000;5(4):473-83. cited
by applicant .
De La Fuente et al., Novel hyaluronan-based nanocarriers for
transmucosal delivery of macromolecules. Macromol Biosci. May 13,
2008;8(5):441-50. cited by applicant .
Delemarre et al., Repopulation of macrophages in popliteal lymph
nodes of mice after liposome-mediated depletion. J Leukoc Biol.
1990;47(3):251-7. cited by applicant .
Demangel et al., Single chain antibody fragments for the selective
targeting of antigens to dendritic cells. Mol Immunol. May
2005;42(8):979-85. Epub Dec. 10, 2004. cited by applicant .
Demello et al., Microscale reactors: nanoscale products. Lab on a
Chip. 2004;4(2):11N-15N. cited by applicant .
Demello, Control and detection of chemical reactions in
microfluidic systems. Nature. 2006;442(7100:394-402. cited by
applicant .
Deming, Facile synthesis of block copolypeptides of defined
architecture. Nature. 1997;390(6658):386-9. cited by applicant
.
Depla et al., Rational design of a multiepitope vaccine encoding
T-lymphocyte epitopes for treatment of chronic hepatitis B virus
infections. J Virol. Jan. 2008;82(1):435-50. Epub Oct. 17, 2007.
cited by applicant .
Derfus et al., Intracellular Delivery of Quantum Dots for Live Cell
Labeling and Organelle Tracking. Adv Mat. 2004;16:961-6. cited by
applicant .
Diethelm-Okita et al., Universal epitopes for human CD4+ cells on
tetanus and diphtheria toxins. J Infect Dis. Mar.
2000;181(3):1001-9. cited by applicant .
Ding et al., Multiepitope peptide-loaded virus-like particles as a
vaccine against hepatitis B virus-related hepatocellular carcinoma.
Hepatology. May 2009;49(5):1492-502. cited by applicant .
Diwan et al., Dose sparing of CpG oligodeoxynucleotide vaccine
adjuvants by nanoparticle delivery. Curr Drug Deliv. Oct.
2004;1(4):405-12. cited by applicant .
Diwan et al., Enhancement of immune responses by co-delivery of a
CpG oligodeoxynucleotide and tetanus toxoid in biodegradable
nanospheres. J Control Release. Dec. 13, 2002;85(1-3):247-62. cited
by applicant .
Donbrow, Ed., Microcapsules and Nanoparticles in Medicine and
Pharmacy. CRC Press, Boca Raton, 1992. cited by applicant .
Dou et al., Development of a macrophage-based nanoparticle platform
for antiretroviral drug delivery. Blood. Oct. 15,
2006;108(8):2827-35. Epub Jun. 29, 2006. Erratum in: Blood. Mar. 1,
2007;109(5):1816. cited by applicant .
Elamanchili et al., "Pathogen-mimicking" nanoparticles for vaccine
delivery to dendritic cells. J Immunother. May-Jun.
2007;30(4):378-95. Abstract only. cited by applicant .
Eldridge et al., Biodegradable microspheres as a vaccine delivery
system. Mol Immunol. 1991;28(3):287-94. cited by applicant .
Farokhzad et al., Drug delivery systems in urology--getting
"smarter". Urology. Sep. 2006;68(3):463-9. cited by applicant .
Farokhzad et al., Impact of nanotechnology on drug delivery. ACS
Nano. Jan. 27, 2009;3(1):16-20. cited by applicant .
Farokhzad et al., Nanoparticle--aptamer bioconjugates for cancer
targeting. Expert Opin Drug Deliv. 2006;3(3):311-24. cited by
applicant .
Farokhzad et al., Nanoparticle-Aptamer Bioconjugates: A New
Approach for Targeting Prostate Cancer Cells. Cancer Research.
2004;64:7668-72. cited by applicant .
Farokhzad et al., Targeted nanoparticle-aptamer bioconjugates for
cancer chemotherapy in vivo. Proc Natl Acad Sci USA.
2006;103(16):6315-20. cited by applicant .
Farr et al., The structure of the sinus wall of the lymph node
relative to its endocytic properties and transmural cell passage.
Am J Anat. 1980;157(3):265-84. cited by applicant .
Feuillet et al., Involvement of Toll-like receptor 5 in the
recognition of flagellated bacteria. Proc Natl Acad Sci U S A. Aug.
15, 2006;103(16):6315-20.Acad Sci U S a. 2006.33):12487-92. Epub
Aug. 4, 2006. cited by applicant .
Fonseca et al., Paclitaxel-loaded PLGA nanoparticles: preparation,
physicochemical characterization and in vitro anti-tumoral
activity. J Control Release. 2002;83(2):273-86. cited by applicant
.
Forslund et al., Nitric oxide-releasing particles inhibit
phagocytosis in human neutrophils. Biochem Biophys Res Commun. Apr.
17, 1997;233(2):492-5. cited by applicant .
Gao et al., In vivo cancer targeting and imaging with semiconductor
quantum dots. Nat Biotechnol. 2004;22(8):969-76. cited by applicant
.
Gao et al., In vivo molecular and cellular imaging with quantum
dots. Curr Op Biotechnol. 2005;16:63-72. cited by applicant .
Garcon et al., Boosting vaccine power. Sci Am. Oct.
2009;301(4):72-9. cited by applicant .
Garrett et al., Novel engineered trastuzumab conformational
epitopes demonstrate in vitro and in vivo antitumor properties
against HER-2/neu. J Immunol. Jun. 1, 2007;178(11):7120-31. cited
by applicant .
Gelperina et al., The potential advantages of nanoparticle drug
delivery systems in chemotherapy of tuberculosis. Am J Respir Crit
Care Med. Dec. 15, 2005;172(12):1487-90. Epub Sep. 8, 2005. cited
by applicant .
Getts et al., Microparticles bearing encephalitogenic peptides
induce T-cell tolerance and ameliorate experimental autoimmune
encephalomyelitis. Nat Biotechnol. Nov. 18, 2012. doi:
10.1038/nbt.2434. [Epub ahead of print]. cited by applicant .
Govender et al., PLGA nanoparticles prepared by nanoprecipitation:
drug loading and release studies of a water soluble drug. J Control
Release. Feb. 1, 1999;57(2):171-85. cited by applicant .
Gref et al., Biodegradable long-circulating polymeric nanospheres.
Science. 1994;263(5153):1600-3. cited by applicant .
Griset et al., Expansile nanoparticles: synthesis,
characterization, and in vivo efficacy of an acid-responsive
polymeric drug delivery system. J Am Chem Soc. Feb. 25,
2009;131(7):2469-71. Epub Jan. 30, 2009. cited by applicant .
Griset, Dissertation entitled: Delivery of Paclitaxel via
pH-Responsive Polymeric Nanoparticles for Prevention of Lung Cancer
and Mesothelioma Recurrence, Ohio State University, 2003. cited by
applicant .
Gu et al., Precise engineering of targeted nanoparticles by using
self-assembled biointegrated block copolymers. Proc Natl Acad Sci U
S A. Feb. 19, 2008;105(7):2586-91. Epub Feb. 13, 2008. cited by
applicant .
Gvili et al., PLGA nanoparticles for DNA vaccination-waiving
complexity and increasing efficiency. Molc Ther. 2006;13:5209.
cited by applicant .
Haas et al., Sequence independent interferon-alpha induction by
multimerized phosphodiester DNA depends on spatial regulation of
Toll-like receptor-9 activation in plasmacytoid dendritic cells.
Immunology. Feb. 2009;126(2):290-8. Epub Nov. 15, 2008. cited by
applicant .
Haddadi, Delivery of rapamycin by PLGA nanoparticles enhances its
suppressive activity on dendritic cells. J Biomed Mat Res A.
2007;84A(4):885-98. cited by applicant .
Haensler et al., Polyamidoamine cascade polymers mediate efficient
transfection of cells in culture. Bioconjugate Chem.
1993;4(5):372-9. cited by applicant .
Hamdy et al., Co-delivery of cancer-associated antigen and
Toll-like receptor 4 ligand in PLGA nanoparticles induces potent
CD8+ T cell-mediated anti-tumor immunity. Vaccine. Sep. 15,
2008;26(39):5046-57. Epub Aug. 3, 2008. cited by applicant .
Hamdy et al., Pharmaceutical analysis of synthetic lipid A-based
vaccine adjuvants in poly (D,L-lactic-co-glycolic acid)
nanoparticle formulations. J Pharm Biomed Anal. Aug. 15,
2007;44(4):914-23. Epub Mar. 19, 2007. cited by applicant .
Hanes et al., Polymer microspheres for vaccine delivery. Pharm
Biotechnol. 1995;6:389-412. cited by applicant .
Hangartner et al., Antiviral immune responses in gene-targeted mice
expressing the immunoglobulin heavy chain of virus-neutralizing
antibodies. Proc Natl Acad Sci USA. 2003;100:12883-88. cited by
applicant .
Harada et al., Supramolecular assemblies of block copolymers in
aqueous media as nanocontainers relevant to biological
applications. Progress Polymer Sci. 2006;31(11):949-82. cited by
applicant .
Harper et al., Efficacy of a bivalent Li virus-like particle
vaccine in prevention of infection with human papillomavirus types
16 and 18 in young women: a randomised controlled trial. Lancet.
2004;364(9447):1757-65. cited by applicant .
Hatsukami et al., Safety and immunogenicity of a nicotine conjugate
vaccine in current smokers. Clin Pharmacol Ther. Nov.
2005;78(5):456-67. cited by applicant .
Hawiger et al., Dendritic cells induce peripheral T cell
unresponsiveness under steady state conditions in vivo. J Exp Med.
2001;194(6):769-79. cited by applicant .
Heeg et al., Structural requirements for uptake and recognition of
CpG oligonucleotides. Int J Med Microbiol. Jan. 2008;298(1-2):33-8.
Epub Aug. 13, 2007. cited by applicant .
Heil et al., Species-specific recognition of single-stranded RNA
via toll-like receptor 7 and 8. Science. Mar. 5,
2004;303(5663):1526-9. Epub Feb. 19, 2004. cited by applicant .
Hemmi et al., A Toll-like receptor recognizes bacterial DNA.
Nature. Dec. 7, 2006;408(6813):740-5. cited by applicant .
Hood et al., Tumor regression by targeted gene delivery to the
neovasculature. Science. Jun. 28, 2002;296(5577):2404-7. cited by
applicant .
Hruby et al., Poly (ethylene oxide)-coated polymide nanoparticles
deradable by glutathione. Colloid Polym Sci. 2007;285:569-74. cited
by applicant .
Johnson et al., Mechanism for rapid self-assembly of block
copolymer nanoparticles. Phys Rev Lett. 2003;91(11):118302.1-4.
cited by applicant .
Jones et al., Polymeric micelles--a new generation of colloidal
drug carriers. Eur J Pharm Biopharm. Sep. 1999;48(2):101-11. cited
by applicant .
Jung et al., Tetanus Toxoid Loaded Nanoparticles from
Sulfobutylated Poly(Vinyl
Alcohol)-Graft-Poly(Lactide-co-Glycolide): Evaluation of Antibody
Response After Oral and Nasal Application in Mice. Pharm Res.
2001;18(3):352-60. cited by applicant .
Junt et al., Subcapsular sinus macrophages in lymph nodes clear
lymph-borne viruses and present them to antiviral B cells. Nature.
2007;450:110-4. Supplemental material. cited by applicant .
Kaba et al., Immune responses of mice with different genetic
backgrounds to improved multiepitope, multitarget malaria vaccine
candidate antigen FALVAC-1A. Clin Vaccine Immunol. Nov.
2008;15(11):1674-83. Epub Sep. 10, 2008. cited by applicant .
Kabanov et al., DNA Complexes with Polycations for the Delivery of
Genetic Material into Cells. Bioconjugate Chem. 1995;6(1):7-20.
cited by applicant .
Kamentsky, Laser scanning cytometry. Methods Cell Biol.
2001;63:51-87. cited by applicant .
Kanchan et al., Interactions of antigen-loaded polylactide
particles with macrophages and their correlation with the immune
response. Biomaterials. Dec. 2007;28(35):5344-57. Epub Sep. 7,
2007. cited by applicant .
Karrer et al., on the key role of secondary lymphoid organs in
antiviral immune responses studied in alymphoplastic (aly/aly) and
spleenless (Hox11(-)/-) mutant mice. J Exp Med.
1997;185(12):2157-70. cited by applicant .
Kelly et al., The Optical Properties of Metal Nanoparticles: The
Influence of Size, Shape, and Dielectric Environment. J Phys Chem
B. 2003;107(3):668-77. cited by applicant .
Khan et al., A systematic bioinformatics approach for selection of
epitope-based vaccine targets. Cell Immunol. Dec.
2006;244(2):141-7. Epub Apr. 16, 2007. cited by applicant .
Kim et al., Enhancement of DNA vaccine potency through
coadministration of CIITA DNA with DNA vaccines via gene gun. J
Immunol. May 15, 2008;180(10):7019-27. cited by applicant .
Kim et al., Inhibition of follicular T-helper cells by CD8(+)
regulatory T cells is essential for self tolerance. Nature. Sep.
16, 2010;467(7313):328-32. cited by applicant .
Kimura et al., Binding of oligoguanylate to scavenger receptors is
required for oligonucleotides to augment NK cell activity and
induce IFN. J Biochem. Nov. 1994;116(5):991-4. cited by applicant
.
Konan et al., Preparation and characterization of sterile sub-200
nm meso-tetra(4-hydroxylphenyl)porphyrin-loaded nanoparticles for
photodynamic therapy. Eur J Pharm Biopharm. Jan. 2003;55(1):115-24.
cited by applicant .
Krieg et al., CpG motifs in bacterial DNA trigger direct B-cell
activation. Nature. 1995;374(6522):546-9. cited by applicant .
Kukowska-Latallo et al., Efficient transfer of genetic material
into mammalian cells using Starburst polyamidoarnine dendrimers.
Proc Natl Acad Sci USA. 1996;93(10):4897-902. cited by applicant
.
Labhasetwar et al., Arterial uptake of biodegradable nanoparticles:
Effect of surface modifications. J Pharm Sci. 1998;87(10):1229-34.
cited by applicant .
Lairmore et al., Human T-lymphotropic virus type 1 peptides in
chimeric and multivalent constructs with promiscuous T-cell
epitopes enhance immunogenicity and overcome genetic restriction. J
Virol. Oct. 1995;69(10):6077-89. cited by applicant .
Lamalle-Bernard et al., Coadsorption of HIV-1 p24 and gp120
proteins to surfactant-free anionic PLA nanoparticles preserves
antigenicity and immunogenicity. J Control Release. Sep. 28,
2006;115(1):57-67. Epub Jul. 13, 2006. cited by applicant .
Langer, Biomaterials in drug delivery and tissue engineering: one
laboratory's experience. Acc Chem Res. 2000;33(2):94-101. cited by
applicant .
Langer, New methods of drug delivery. Science.
1990;249(4976):1527-33. cited by applicant .
Langer, Selected advances in drug delivery and tissue engineering.
J Control Release. 1999;62:7-11. cited by applicant .
Lee et al., Adaptations of nanoscale viruses and other protein
cages for medical applications. Nanomedicine. Sep.
2006;2(3):137-49. cited by applicant .
Leopold et al., Fluorescent virions: dynamic tracking of the
pathway of adenoviral gene transfer vectors in living cells. Hum
Gene Ther. 1998;9(3):367-78. cited by applicant .
Leucuta et al., Albumin microspheres as a drug delivery system for
epirubicin: pharmaceutical, pharmacokinetic and biological aspects.
Int J Phar. 1988;41:213-7. cited by applicant .
Liang et al., Activation of human B cells by phosphorothioate
oligodeoxynucleotides. J Clin Invest. Sep. 1, 1996;98(5):1119-29.
cited by applicant .
Liang et al., Paclitaxel-Loaded Poly(.gamma.-glutamic
acid)-poly(lactide) Nanoparticles as a Targeted Drug Delivery
System against Cultured HepG2 Cells. Bioconjug Chem. Mar.-Apr.
2006;17(2):291-9. E-pub ahead of print. E-pub version. cited by
applicant .
Lim et al., A Self-Destroying Polycationic Polymer: Biodegradable
Poly(4-hydroxy-L- proline ester). J Am Chem Soc.
1999;121(24):5633-9. cited by applicant .
Lim et al., Cationic hyperbranched poly(amino ester): a novel class
of DNA condensing molecule with cationic surface, biodegradable
three-dimensional structure, and tertiary amine groups in the
interior. J Am Chem Soc. Mar. 14, 2001;123(10):2460-1. cited by
applicant .
Lin et al., Well-Ordered Mesoporous Silica Nanoparticles as Cell
Markers. Chem Mater. 2005;17:4570-3. cited by applicant .
Lindblad, Aluminium compounds for use in vaccines. Immunol Cell
Biol. Oct. 2004;82(5):497-505. cited by applicant .
Lipford et al., Bacterial DNA as immune cell activator. Trends
Microbiol. Dec. 1998;6(12):496-500. cited by applicant .
Lipford et al., CpG-containing synthetic oligonucleotides promote B
and cytotoxic T cell responses to protein antigen: a new class of
vaccine adjuvants. Eur J Immunol. Sep. 1997;27(9):2340-4. cited by
applicant .
Livingston et al., A rational strategy to design multiepitope
immunogens based on multiple Thlymphocyte epitopes. J Immunol. Jun.
1, 2002;168(11):5499-506. cited by applicant .
Lloyd, Disulphide reduction in lysosomes. The role of cysteine.
Biochem J. Jul. 1, 1986;237(1):271-2. cited by applicant .
Lonnberg, Solid-phase synthesis of oligonucleotide conjugates
useful for delivery and targeting of potential nucleic acid
therapeutics. Bioconjug Chem. Jun. 2009;20(6):1065-94. cited by
applicant .
Low et al., Folate receptor-targeted drugs for cancer and
inflammatory diseases. Adv Drug Deliv Rev. 2004;56(8):1055-8. cited
by applicant .
Ludewig et al., Induction of optimal anti-viral neutralizing B cell
responses by dendritic cells requires transport and release of
virus particles in secondary lymphoid organs. Eur J Immunol.
2000;30(1):185-96. cited by applicant .
Malyala et al., Enhancing the therapeutic efficacy of CpG
oligonucleotides using biodegradable microparticles. Adv Drug Deliv
Rev. Mar. 28, 2009;61(3):218-25. Epub Jan. 11, 2009. cited by
applicant .
Malyala et al., The potency of the adjuvant, CpG oligos, is
enhanced by encapsulation in PLG microparticles. J Pharm Sci. Mar.
2008;97(3):1155-64. cited by applicant .
Manolova et al., Nanoparticles target distinct dendritic cell
populations according to their size. Eur J Immunol.
2008;38:1404-13. cited by applicant .
Martin et al., A vector-based minigene vaccine approach results in
strong induction of T-cell responses specific of hepatitis C virus.
Vaccine. May 12, 2008;26(20):2471-81. Epub Apr. 1, 2008. cited by
applicant .
Martinez-Pomares et al., Antigen presentation the macrophage way.
Cell. Nov. 16, 2007;131(4):641-3. cited by applicant .
Mathiowitz et al., Novel microcapsules for delivery systems.
Reactive Polymers. 1987;6:275-83. cited by applicant .
Mathiowitz et al., Polyanhydride Microspheres as Drug Carriers I.
Hot-Melt Microencapsulation. J Control Release. 1987;5:13-22. cited
by applicant .
Mathiowitz et al., Polyanhydride Microspheres as Drug Carriers. II
. . . Microencapsulation by Solvent Removal. J Appl Polymer Sci.
1988;35:755-74. cited by applicant .
Mattheakis et al., Optical coding of mammalian cells using
semiconductor quantum dots. Anal Biochem. 2004;327(2):200-8. cited
by applicant .
Maye et al., Comparison of the phagocytosis of two types of
cyclosporin (SDZ OXL 400 and SDZ IMM 125) by alveolar macrophages
from hamsters. Cell Biol Toxicol. Dec. 1998;14(6):411-8. cited by
applicant .
McSorley et al., Bacterial flagellin is an effective adjuvant for
CD4+ T cells in vivo. J Immunol. Oct. 1, 2002;169(7):3914-9. cited
by applicant .
Meister et al., Mechanisms of gene silencing by double-stranded
RNA. Nature. 2004;431(7006):343-9. cited by applicant .
Mempel et al., T-cell priming by dendritic cells in lymph nodes
occurs in three distinct phases. Nature. 2004;427(6970):154-9.
cited by applicant .
Metelitsa et al., Antidisialoganglioside/granulocyte
macrophage-colonystimulating factor fusion protein facilitates
neutrophil antibody-dependent cellular cytotoxicity and depends on
Fc.gamma.RII (CD32) and Mac-1 (CD11b/CD18) for enhanced effector
cell adhesion and azurophil granule exocytosis. Blood.
2002;99(11):4166-73. cited by applicant .
Michiels et al., Patent exemption for clinical trials: current
status of the Bolar-type provisions in Europe. Life Sciences
Intellectual Property Review 2008. Lavoix. www.worldipreview.com.
2008:68-70. cited by applicant .
Miyara et al., Therapeutic approaches to allergy and autoimmunity
based on FoxP3+ regulatory T-cell activation and expansion. J
Allergy Clin Immunol. Apr. 2009;123(4):749-55. cited by applicant
.
Moghimi et al., Long-circulating and target-specific nanoparticles:
theory to practice. Pharmacol Rev. 2001;53(2):283-318. cited by
applicant .
Moghimi et al., Nanomedicine: current status and future prospects.
FASEB J. Mar. 2005;19(3):311-30. cited by applicant .
Mulligan, The basic science of gene therapy.
Science.1993;260(5110):926-32. cited by applicant .
Murray et al., Synthesis and characterization of monodisperse
nanocrystals and close-packed nanocrystal assemblies. Ann Rev Mat
Sci. 2000;30:545-610. cited by applicant .
Nakase et al., Biodegradable microspheres targeting mucosal
immune-regulating cells: new approach for treatment of inflammatory
bowel disease. J Gastroenterol. Mar. 2003;38 Suppl 15:59-62. cited
by applicant .
Nielsen et al., Therapeutic efficacy of anti-ErbB2 immunoliposomes
targeted by a phage antibody selected for cellular endocytosis.
Biochim Biophys Acta. Aug. 19, 2002;1591(1-3):109-118. cited by
applicant .
Nikou et al., A HER-2/neu peptide admixed with PLA microspheres
induces a Th1-biased immune response in mice. Biochim Biophys Acta.
Sep. 15, 2005;1725(2):182-9. cited by applicant .
Notter et al., Targeting of a B7-1 (CD80) immunoglobulin G fusion
protein to acute myeloid leukemia blasts increases their
costimulatory activity for autologous remission T cells. Blood.
2001;97(10):3138-45. cited by applicant .
Ochsenbein et al., Control of early viral and bacterial
distribution and disease by natural antibodies. Science.
1999;286(5447):2156-9. cited by applicant .
Ochsenbein et al., Protective T cell-independent antiviral antibody
responses are dependent on complement. J Exp Med.
1999;190(8):1165-74. cited by applicant .
Oh et al., CD4+CD25+ regulatory T cells in autoimmune arthritis.
Immunol Rev. Jan. 2010;233(1):97-111. cited by applicant .
Okada et al., Antigen-engaged B cells undergo chemotaxis toward the
T zone and form motile conjugates with helper T cells. PLoS Biol.
2005;3(6):e150. 1047-61. cited by applicant .
Olivier et al., Synthesis of pegylated immunonanoparticles. Pharm
Res. Aug. 2002;19(8):1137-43. cited by applicant .
Ong et al., Redox-triggered contents release from liposomes. J Am
Chem Soc. Nov. 5, 2008;130(44):14739-44. Epub Oct. 8, 2008. cited
by applicant .
O'Sullivan et al., Truncation analysis of several DR binding
epitopes. J Immunol. Feb. 15, 1991;146(4):1240-6. cited by
applicant .
Paoletti et al. eds., Vaccines: from Concept to Clinic. A Guide to
the Development and Clinical Testing of Vaccines for Human Use.
1999 CRC Press. 210 pages. cited by applicant .
Pape et al., The humoral immune response is initiated in lymph
nodes by B cells that acquire soluble antigen directly in the
follicles. Immunity. 2007;26(4):491-502. cited by applicant .
Pasqualini et al., Aminopeptidase N is a receptor for tumor-homing
peptides and a target for inhibiting angiogenesis. Cancer Res.
2000;60(3):722-7. cited by applicant .
Patri et al., Synthesis and in Vitro Testing of J591
Antibody--Dendrimer Conjugates for Targeted Prostate Cancer
Therapy. Bioconj Chem. 2004;15:1174-81. cited by applicant .
Pellegrino et al., On the development of colloidal nanoparticles
towards multifunctional structures and their possible use for
biological applications. Small. 2005;1(1):48-63 cited by applicant
.
Phillips et al., Enhanced antibody response to liposome-associated
protein antigens: preferential stimulation of IgG2a/b production.
Vaccine. 1992;10(3):151-8. cited by applicant .
Pimentel et al., Peptide nanoparticles as novel immunogens: design
and analysis of a prototypic severe acute respiratory syndrome
vaccine. Chem Biol Drug Des. Jan. 2009;73(1):53-61. cited by
applicant .
Pitaksuteepong, Nanoparticles: A vaccine adjuvant for subcutaneous
administration. Naresuan University J. 2005;13(2):53-62. cited by
applicant .
Popielarski et al., A nanoparticle-based model delivery system to
guide the rational design of gene delivery to the liver. 2. In
vitro and in vivo uptake results. Bioconjug Chem. Sep.-Oct.
2005;16(5):1071-80. cited by applicant .
Purcell et al., Dissecting the role of peptides in the immune
response: theory, practice and the application to vaccine design. J
Pept Sci. May 2003;9(5):255-81. cited by applicant .
Purcell et al., More than one reason to rethink the use of peptides
in vaccine design. Nat Rev Drug Discov. May 2007;6(5):404-14. cited
by applicant .
Qi et al., Extrafollicular activation of lymph node B cells by
antigen-bearing dendritic cells. Science. 2006;312(5780):1672-6.
cited by applicant .
Qiu et al., PLA-coated gold nanoparticles for the labeling of PLA
biocarriers. Chem Mater. 2004;16:850-6. cited by applicant .
Quintanar-Guerrero et al., Preparation Techniques and Mechanisms of
Formation of Biodegradable Nanoparticles from Preformed Polymers.
Drug Dev Industrial Pharmacy. 1998;24(12):1113-28. cited by
applicant .
Raman et al., Peptide Based Nanoparticles as a Platform for Vaccine
Design.
http://www.nsti.org/Nanotech2005/showabstract.html?absno=637. 2005.
Abstract Only. cited by applicant .
Reddy et al., Exploiting lymphatic transport and complement
activation in nanoparticle vaccines. Nat Biotech.
2007;25(10):1159-64. cited by applicant .
Reif et al., Balanced responsiveness to chemoattractants from
adjacent zones determines B-cell position. Nature.
2002;416(6876):94-9. cited by applicant .
Reis et al., Nanoencapsulation I. Methods for preparation of
drug-loaded polymeric nanoparticles. Nanomedicine. 2006;2:8-21.
cited by applicant .
Robbins et al., Stable expression of shRNAs in human CD34+
progenitor cells can avoid induction of interferon responses to
siRNAs in vitro. Nature Biotechnology. 2006;24(5):566-71. cited by
applicant .
Roman et al., Immunostimulatory DNA sequences function as T
helper-1-promoting adjuvants. Nat Med. Aug. 1997;3(8):849-54. cited
by applicant .
Rossbacher et al, The B cell receptor itself can activate
complement to provide the complement receptor 1/2 ligand required
to enhance B cell immune responses in vivo. J Exp Med.
2003;198(4):591-602. cited by applicant .
Salmeron et al., Encapsulation Study of 6-Methylprednisolone in
Lipid Microspheres. Drug Develop Indust Pharm. 1997;23(2):133-6.
cited by applicant .
Samuel et al, Polymeric nanoparticles for targeted delivery of
therapeutic vaccines to dendritic cells. Proc Intl Conf on MEMS,
NANO and Smart Sys. Jul. 2003;20-23:242-6. cited by applicant .
Scardino et al., A polyepitope DNA vaccine targeted to Her-2/ErbB-2
elicits a broad range of human and murine CTL effectors to protect
against tumor challenge. Cancer Res. Jul. 15, 2007;67(14):7028-36.
cited by applicant .
Schultz et al., Single-target molecule detection with nonbleaching
multicolor optical immunolabels. Proc Natl Acad Sci USA.
2000;97(3):996-1001. cited by applicant .
Schultz, Plasmon resonant particles for biological detection. Curr
Op Biotechnol. 2003;14:13-22. cited by applicant .
Senger et al., Identification of B-cell epitopes on virus-like
particles of cutaneous alpha-human papillomaviruses. J Virol. Dec.
2009;83(24):12692-701. Epub Sep. 30, 2009. cited by applicant .
Shahiwala et al., Nanocarriers for systemic and mucosal vaccine
delivery. Recent Pat Drug Deliv Formul. 2007;1(1):1-9. cited by
applicant .
Sharma et al., Pharmaceutical aspects of intranasal delivery of
vaccines using particulate systems. J Pharm Sci. Mar.
2009;98(3):812-43. cited by applicant .
Shen et al., Enhanced and prolonged cross-presentation following
endosomal escape of exogenous antigens encapsulated in
biodegradable nanoparticles. Immunol. 2006;117:78-88. cited by
applicant .
Shestopalov et al., Multi-step synthesis of nanoparticles performed
on millisecond time scale in a microfluidic droplet-based system.
Lab Chip. 2004;4(4):316-21. cited by applicant .
Shiow et al., CD69 acts downstream of interferon-alpha/beta to
inhibit S1P1 and lymphocyte egress from lymphoid organs. Nature.
Mar. 23, 2006;440(7083):540-4. Epub Mar. 8, 2006. cited by
applicant .
Singh et al., Anionic microparticles are a potent delivery system
for recombinant antigens from Neisseria meningitidis serotype B. J
Pharm Sci. Feb. 2004;93(2):273-82. cited by applicant .
Singh et al., Cationic microparticles are an effective delivery
system for immune stimulatory cpG DNA. Pharm Res. Oct.
2001;18(10):1476-9. cited by applicant .
Singh et al., Nanoparticles and microparticles as vaccine-delivery
systems. Expert Rev Vaccines. Oct. 2007;6(5):797-808. cited by
applicant .
Sondel et al., Preclinical and clinical development of
immunocytokines. Curr Opin Investig Drugs. 2003;4(6):696-700. cited
by applicant .
Sonehara et al., Hexamer palindromic oligonucleotides with 5'-CG-3'
motif(s) induce production of interferon. J Interferon Cytokine
Res. Oct. 1996;16(10):799-803. cited by applicant .
Srinivasan et al., Prediction of class I T-cell epitopes: evidence
of presence of immunological hot spots inside antigens.
Bioinformatics. Aug. 4, 2004;20 Suppl 1:i297-302. cited by
applicant .
Stivaktakis et al., Immune responses in mice of beta-galactosidase
adsorbed or encapsulated in poly(lactic acid) and
poly(lactic-co-glycolic acid) microspheres. J Biomed Mater Res A.
Jun. 1, 2005;73(3):332-8. cited by applicant .
Stivaktakis et al., PLA and PLGA microspheres of
beta-galactosidase: Effect of formulation factors on protein
antigenicity and immunogenicity. J Biomed Mater Res A. Jul. 1,
2004;70(1):139-48. cited by applicant .
Storm et al., Surface Modification of Nanoparticles to Oppose
Uptake by the Mononuclear Phagocyte System. Adv Drug Deliv Rev.
1995;17:31-48. cited by applicant .
Suri et al., Nanotechnology-based drug delivery systems. J Occup
Med Toxicol. Dec. 1, 2007;2:16. cited by applicant .
Tabata et al., Macrophage activation through phagocytosis of poly
(L-lactic acid) microspheres containing an immunomodulatory agent.
1989;7(2):79-86. Abstract only. cited by applicant .
Tabata et al., Protein precoating of polylactide microspheres
containing a lipophilic immunopotentiator for enhancement of
macrophage phagocytosis and activation. Pharm Res. Apr.
1989;6(4):296-301. cited by applicant .
Tang et al., Adenovirus hexon T-cell epitope is recognized by most
adults and is restricted by HLA DP4, the most common class II
allele. Gene Ther. Sep. 2004;11(18):1408-15. cited by applicant
.
Tang et al., In Vitro Gene Delivery by Degraded Polyamidoamine
Dendrimers. Bioconjugate Chem. 1996;7:703-14. cited by applicant
.
Tarlinton et al., Antigen to the node: B cells go native. Immunity.
Apr. 2007;26(4):388-90. cited by applicant .
Taylor et al., Macrophage receptors and immune recognition. Annu
Rev Immunol. 2005;23:901-44. cited by applicant .
Timmerman, Carrier protein conjugate vaccines: the "missing link"
to improved antibody and CTL responses? Hum Vaccin. Mar.
2009;5(3):181-3. Epub Mar. 24, 2009. cited by applicant .
Tissot et al., Versatile virus-like particle carrier for epitope
based vaccines. PLoS One. Mar. 23, 2010;5(3):e9809. cited by
applicant .
Tomai et al., Resiquimod and other immune response modifiers as
vaccine adjuvants. Expert Rev Vaccines. Oct. 2007;6(5):835-47.
cited by applicant .
Tong et al., Ring-opening polymerization-mediated controlled
formulation of polylactide-drug nanoparticles. J Am Chem Soc. Apr.
8, 2009;131(13):4744-54. E-pub Mar. 12, 2009. cited by applicant
.
Trindade et al., Nanocrystalline Semiconductors: Synthesis,
Properties, and Perspectives. Chem Mat. 2001;13(11):3843-58. cited
by applicant .
Uhrich et al., Polymeric Systems for Controlled Drug Release. Chem
Rev. 1999;99(11):3181-98. cited by applicant .
Unkeless et al., Structure and function of human and murine
receptors for IgG. Annu Rev Immunol. 1998;6:251-81. cited by
applicant .
Uwatoku et al., Application of Nanoparticle Technology for the
Prevention of Restenosis After Balloon Injury in Rats. Circ Res.
2003;92(7):e62-9. cited by applicant .
Van Broekhoven et al., Targeting dendritic cells with
antigen-containing liposomes: a highly effective procedure for
induction of antitumor immunity and for tumor immunotherapy. Cancer
Res.Jun. 15, 2004;64(12):4357-65. cited by applicant .
Vascotto et al., Antigen presentation by B lymphocytes: how
receptor signaling directs membrane trafficking. Curr Opin Immunol.
2007;19(1):93-8. cited by applicant .
Vauthier et al., Design aspects of poly(alkylcyanoacrylate)
nanoparticles for drug delivery. J Drug Target. Dec.
2007;15(10):641-63. cited by applicant .
Vila et al., Regulatory T cells and autoimmunity. Curr Opin
Hematol. Jul. 2009;16(4):274-9. cited by applicant .
Vita et al., The immune epitope database 2.0. Nucleic Acids Res.
Jan. 2010;38(Database issue):D854-62. Epub Nov. 11, 2009. cited by
applicant .
Vollmer et al., Characterization of three CpG oligodeoxynucleotide
classes with distinct immunostimulatory activities. Eur J Immunol.
Jan. 2004;34(1):251-62. cited by applicant .
Von Andrian et al., Homing and cellular traffic in lymph nodes. Nat
Rev Immunol. 2003;3(11):867-78. cited by applicant .
Weber et al., T cell epitope: friend or foe? Immunogenicity of
biologics in context. Adv Drug Deliv Rev. Sep. 30,
2009;61(11):965-76. Epub Jul. 18, 2009. cited by applicant .
Wessels et al., Studies of group B streptococcal infection in mice
deficient in complement component C3 or C4 demonstrate an essential
role for complement in both innate and acquired immunity. Proc Natl
Acad Sci USA. 1995;92(25):11490-4. cited by applicant .
Whelan et al., Efficient recovery of infectious vesicular
stomatitis virus entirely from cDNA clones. Proc Natl Acad Sci USA.
1995;92(18):8388-92. cited by applicant .
Wu et al., A novel chitosan CpG nanoparticle regulates cellular and
humoral immunity of mice.Biomed Environ Sci. Apr. 2006;19(2):87-95.
cited by applicant .
Wu et al., Resiquimod: a new immune response modifier with
potential as a vaccine adjuvant for Th1 immune responses. Antiviral
Res. Nov. 2004;64(2):79-83. cited by applicant .
Yang et al., Tumor necrosis factor alpha blocking peptide loaded
PEG-PLGA nanoparticles: preparation and in vitro evaluation. Int J
Pharm. Feb. 22, 2007;331(1):123-32. cited by applicant .
Yang, Imaging of vascular gene therapy. Radiology. 2003;228:36-49.
cited by applicant .
Yoo et al., in vitro and In vivo anti-tumor activities of
nanoparticles based on doxorubicin--PLGA conjugates. J Control
Release. 2000;68(3):419-31. cited by applicant .
Yu et al., Potent CpG oligonucleotides containing phosphodiester
linkages: in vitro and in vivo immunostimulatory properties.
Biochem Biophys Res Commun. Sep. 13, 2002;297(1):83-90. cited by
applicant .
Yuan et al., Intranasal immunization with chitosan/pCETP
nanoparticles inhibits atherosclerosis in a rabbit model of
atherosclerosis. Vaccine. Jul. 4, 2008;26(29-30):3727-34. Epub May
16, 2008. cited by applicant .
Zauner et al., Polylysine-based transfection systems utilizing
receptor-mediated delivery. Adv Drug Del Rev. 1998;30:97-113. cited
by applicant .
Zhang et al., A comparative study of the antigen-specific immune
response induced by co-delivery of CpG ODN and antigen using fusion
molecules or biodegradable microparticles. J Pharm Sci. Dec.
2007;96(12):3283-92. cited by applicant .
Zhang et al., Nanoparticles of poly(lactide)/vitamin E TPGS
copolymer for cancer chemotherapy: synthesis, formulation,
characterization and in vitro drug release. Biomaterials. Jan.
2006;27(2):262-70. cited by applicant .
Zhang-Hoover et al., Tolerogenic APC generate CD8+ T regulatory
cells that modulate pulmonary interstitial fibrosis. J Immunol.
Jan. 1, 2004;172(1):178-85. cited by applicant .
Zheng et al., Highly fluorescent, water-soluble, size-tunable gold
quantum dots. Phys Rev Lett. 2004;93(7):077402.1-4. cited by
applicant .
Zheng et al., How antigen quantity and quality determine T-cell
decisions in lymphoid tissue. Mol Cell Biol. Jun.
2008;28(12):4040-51. Epub Apr. 21, 2008. cited by applicant .
Zhou et al., Investigation on a novel core-coated microspheres
protein delivery system. J Control Release. Jul. 10,
2001;75(1-2):27-36. cited by applicant .
Zhu et al., T cell epitope mapping of ragweed pollen allergen
Ambrosia artemisiifolia (Amb a 5) and Ambrosia trifida (Amb t 5)
and the role of free sulfhydryl groups in T cell recognition. J
Immunol. Nov. 15, 1995;155(10):5064-73. cited by applicant .
Zwiorek et al., Delivery by cationic gelatin nanoparticles strongly
increases the immunostimulatory effects of CpG oligonucleotides.
Pharm Res. Mar. 2008;25(3):551-62. Epub Oct. 3, 2007. cited by
applicant .
U.S. Appl. No. 13/560,943, filed Jul. 27, 2012, Gao et al. cited by
applicant .
Extended European Search Report for Application No. EP 12819411.5
dated Mar. 18, 2015. cited by applicant .
Stano et al., PPS nanoparticles as versatile delivery system to
induce systemic and broad mucosal immunity after intranasal
administration. Vaccine. Jan. 17, 2011;29(4):804-12. doi:
10.1016/j.vaccine.2010.11.010. Epub Nov. 19, 2010. cited by
applicant .
EP 12819411.5, Mar. 18, 2015, Extended European Search Report.
cited by applicant .
U.S. Appl. No. 14/658,040, filed Mar. 13, 2015, Zepp et al. cited
by applicant .
U.S. Appl. No. 14/717,451, filed May 20, 2015, Ilyinskii et al.
cited by applicant .
U.S. Appl. No. 15/050,397, filed Feb. 22, 2016, Fraser et al. cited
by applicant .
U.S. Appl. No. 14/810,418, filed Jul. 27, 2015, Fraser et al. cited
by applicant .
U.S. Appl. No. 14/802,260, filed Jul. 17, 2015, Altreuter et al.
cited by applicant .
U.S. Appl. No. 14/810,427, filed Jul. 27, 2015, Fraser et al. cited
by applicant .
U.S. Appl. No. 15/061,096, filed Mar. 4, 2016, Fraser et al. cited
by applicant .
U.S. Appl. No. 14/810,442, filed Jul. 27, 2015, Fraser et al. cited
by applicant .
U.S. Appl. No. 14/10,450, filed Jul. 27, 2015, Fraser et al. cited
by applicant .
U.S. Appl. No. 14/810,457, filed Jul. 27, 2015, Kishimoto et al.
cited by applicant .
U.S. Appl. No. 15/061,204, filed Mar. 4, 2016, Kishimoto et al.
cited by applicant .
U.S. Appl. No. 14/810,466, filed Jul. 27, 2015, Kishimoto et al.
cited by applicant .
U.S. Appl. No. 14/810,472, filed Jul. 27, 2015, Fraser et al. cited
by applicant .
U.S. Appl. No. 14/810,476, filed Jul. 27, 2015, Maldonado. cited by
applicant .
U.S. Appl. No. 14/269,047, filed May 2, 2015, Maldonado et al.
cited by applicant .
U.S. Appl. No. 14/269,056, filed May 2, 2014, Maldonato et al.
cited by applicant .
U.S. Appl. No. 14/742,583, filed Jun. 17, 2015, Kishimoto. cited by
applicant .
U.S. Appl. No. 14/751,106, filed Jun. 25, 2015, Kishimoto et al.
cited by applicant .
U.S. Appl. No. 14/846,949, filed Sep. 7, 2015, Kishimoto. cited by
applicant .
U.S. Appl. No. 14/846,952, filed Sep. 7, 2015, Kishimoto. cited by
applicant .
U.S. Appl. No. 14/846,958, filed Sep. 7, 2015, Kishimoto. cited by
applicant .
U.S. Appl. No. 14/846,964, filed Sep. 7, 2015, Kishimoto. cited by
applicant .
U.S. Appl. No. 14/934,132, filed Nov. 5, 2015, Griset et al. cited
by applicant .
U.S. Appl. No. 14/934,135, filed Nov. 5, 2015, Griset et al. cited
by applicant .
U.S. Appl. No. 14/846,967, filed Sep. 7, 2015, Kishimoto. cited by
applicant .
U.S. Appl. No. 15/456,520, filed Mar. 11, 2017, Johnston. cited by
applicant .
Robbins et al., Fabricated Nanoparticles with Cross Validation
Using a Humanized Mouse Model. Nanomed Nanotech Biol Med. 2015.
Accepted manuscript. doi: 10.1016/j.nano.2014.11.010. 36 pages.
cited by applicant .
Thomas et al., Engineering complement activation on polypropylene
sulfide vaccine nanoparticles. Biomaterials. Mar.
2011;32(8):2194-203. doi: 10.1016/j.biomaterials.2010.11.037. cited
by applicant .
Wong et al., Cutting edge: antigen-independent CD8 T cell
proliferation. J Immunol. May 15, 2001;166(10):5864-8. cited by
applicant .
[No Author Listed] Drug Delivery System. May 2007;22(3):289. cited
by applicant .
Akagi et al., Development of vaccine adjuvants using polymeric
nanoparticles and their potential applications for anti-HIV
vaccine. Yakugaku Zasshi. 2007;127(2):307-17. cited by applicant
.
Anderson et al., Cytotoxic T cells. J Invest Dermatol.
2006;126:32-41. DOI:10.1038/sj.jid5700001. cited by applicant .
Ma et al., Enhanced presentation of MHC Class Ia, Ib and Class
II--restricted peptides encapsulated in biodegradable
nanoparticles: a promising strategy for tumor immunotherapy. J
Transl Med. Mar. 31, 2011;9(34):1-10.
https://doi.org/10.1186/1479-5876-9-34. cited by applicant .
U.S. Appl. No. 12/788,260, filed May 26, 2010, Zepp et al. cited by
applicant .
U.S. Appl. No. 13/948,129, filed Jul. 22, 2013, Zepp et al. cited
by applicant .
U.S. Appl. No. 14/273,099, filed May 8, 2014, Zepp et al. cited by
applicant .
U.S. Appl. No. 12/788,261, filed May 26, 2010, Lipford et al. cited
by applicant .
U.S. Appl. No. 14/138,601, filed Dec. 23, 2013, Zepp et al. cited
by applicant .
U.S. Appl. No. 12/862,076, filed Aug. 24, 2010, Fraser et al. cited
by applicant .
U.S. Appl. No. 13/116,453, filed May 26, 2011, Bratzler et al.
cited by applicant .
U.S. Appl. No. 12/764,569, filed Apr. 21, 2010, Lipford et al.
cited by applicant .
U.S. Appl. No. 13/116,488, filed May 26, 2011, Bratzler et al.
cited by applicant .
U.S. Appl. No. 13/116,542, filed May 26, 2011, Ilyinskii et al.
cited by applicant .
U.S. Appl. No. 13/116,556, filed May 26, 2011, Bratzler et al.
cited by applicant .
U.S. Appl. No. 13/289,211, filed Nov. 4, 2011, Zepp et al. cited by
applicant .
U.S. Appl. No. 13/428,340, filed Mar. 23, 2012, Altreuter et al.
cited by applicant .
U.S. Appl. No. 13/458,021, filed Apr. 27, 2012, Fraser et al. cited
by applicant .
U.S. Appl. No. 13/458,980, filed Apr. 27, 2012, Altreuter et al.
cited by applicant .
U.S. Appl. No. 13/458,067, filed Apr. 27, 2012, Fraser et al. cited
by applicant .
U.S. Appl. No. 13/457,994, filed Apr. 27, 2012, Fraser et al. cited
by applicant .
U.S. Appl. No. 13/457,999, filed Apr. 27, 2012, Fraser et al. cited
by applicant .
U.S. Appl. No. 13/457,977, filed Apr. 27, 2012, Kishimoto et al.
cited by applicant .
U.S. Appl. No. 13/457,936, filed Apr. 27, 2012, Kishimoto et al.
cited by applicant .
U.S. Appl. No. 13/458,220, filed Apr. 27, 2012, Fraser et al. cited
by applicant .
U.S. Appl. No. 14/161,660, filed Jan. 22, 2014, Maldonado. cited by
applicant .
U.S. Appl. No. 14/269,047, filed May 2, 2014, Maldonado et al.
cited by applicant .
U.S. Appl. No. 14/296,204, filed Jun. 4, 2014, Maldonado et al.
cited by applicant .
U.S. Appl. No. 14/269,048, filed May 2, 2014, Maldonado. cited by
applicant .
U.S. Appl. No. 14/269,054, filed May 2, 2014, Maldonado. cited by
applicant .
U.S. Appl. No. 14/269,058, filed May 2, 2014, Kishimoto. cited by
applicant .
U.S. Appl. No. 14/269,056, filed May 2, 2014, Maldonado et al.
cited by applicant .
U.S. Appl. No. 14/269,042, filed May 2, 2014, Kishimoto et al.
cited by applicant .
PCT/US2012/048670, Feb. 27, 2013, International Search Report and
Written Opinion. cited by applicant .
PCT/US2012/048670, Feb. 13, 2014, International Preliminary Report
on Patentability. cited by applicant.
|
Primary Examiner: Kim; Yunsoo
Attorney, Agent or Firm: Wolf, Greenfield & Sacks,
P.C.
Parent Case Text
RELATED APPLICATIONS
This application claims the benefit under 35 U.S.C. .sctn. 119 of
U.S. provisional application 61/513,496, 61/513,526 and 61/513,527,
each filed Jul. 29, 2011, the entire contents of each of which are
incorporated herein by reference.
Claims
What is claimed is:
1. A method consisting essentially of: identifying a human subject
in need of a humoral and cytotoxic T lymphocyte (CTL) immune
response to a first protein, and administering to the human subject
a composition comprising one or more antigens that are associated
with a disease, disorder or condition, wherein at least one antigen
is the first protein coupled to a population of synthetic
nanocarriers; wherein the first protein comprises at least one
humoral epitope and at least one MHC Class I-restricted epitope
that are not the same epitope, wherein the population of synthetic
nanocarriers does not comprise a saponin-cholesterol adjuvant, and
wherein the mean of a particle size distribution obtained using
dynamic light scattering of the population of synthetic
nanocarriers is a maximum dimension of from 20 nm to 250 nm, and
administering to the human subject a single adjuvant.
2. A method, consisting essentially of: administering to a human
subject a composition comprising one or more antigens that are
associated with a disease, disorder or condition, wherein at least
one antigen is a first protein coupled to a population of synthetic
nanocarriers; wherein the first protein comprises at least one
humoral epitope and at least one MHC Class I-restricted epitope
that are not the same epitope, wherein the population of synthetic
nanocarriers does not comprise a saponin-cholesterol adjuvant,
wherein the mean of a particle size distribution obtained using
dynamic light scattering of the population of synthetic
nanocarriers is a maximum dimension of from 20 nm to 250 nm, and
wherein the composition is administered according to a vaccination
regimen that achieves immunity via both the humoral and cytotoxic T
lymphocyte arms of the immune system, and administering to the
human subject a single adjuvant.
3. A method, consisting essentially of: administering to a human
subject a composition comprising one or more antigens that are
associated with a disease, disorder or condition, wherein at least
one antigen is a first protein coupled to a population of synthetic
nanocarriers; wherein the first protein comprises at least one
humoral epitope and at least one MHC Class I-restricted epitope
that are not the same epitope, wherein the population of synthetic
nanocarriers does not comprise a saponin-cholesterol adjuvant,
wherein the mean of a particle size distribution obtained using
dynamic light scattering of the population of synthetic
nanocarriers is a maximum dimension of from 20 nm to 250 nm, and
wherein the composition is administered according to a protocol
that was previously shown to result in a humoral and CTL immune
response specific to the first protein in one or more test
subjects, and administering to the human subject a single
adjuvant.
4. The method of any one of claims 1-3, wherein the composition is
administered in an amount effective to generate a humoral and CTL
immune response to the first protein.
5. The method of any one of claims 1-3, wherein the composition
further comprises the single adjuvant.
6. The method of any one of claims 1-3, wherein the adjuvant is a
stimulator or agonist of pattern recognition receptors, mineral
salt, alum, MPL.RTM. (AS04), AS15, saponin, QS-21, Quil-A, ISCOMs,
ISCOMATRIX.TM., MF59.TM., Montanide.RTM. ISA 51, Montanide.RTM. ISA
720, AS02, a liposome or liposomal formulation, AS01, synthesized
or specifically prepared microparticles and microcarriers,
bacteria-derived outer membrane vesicle of N. gonorrhoeae or
Chlamydia trachomatis, chitosan particles, depot-forming agent,
Pluronic.RTM. block co-polymer, specifically modified or prepared
peptide, muramyl dipeptide, aminoalkyl glucosaminide 4-phosphate,
RC529, bacterial toxoid, toxin fragment, agonist of Toll-Like
Receptors 2, 3, 4, 5, 7, 8, or 9; adenine derivative;
immunostimulatory DNA; immunostimulatory RNA; imidazoquinoline
amine, imidazopyridine amine, 6,7-fused cycloalkylimidazopyridine
amine, 1,2-bridged imidazoquinoline amine; imiquimod; resiquimod;
agonist for DC surface molecule CD40; type I interferon; poly I:C;
bacterial lipopolysaccharide (LPS); VSV-G; HMGB-1; flagellin;
immunostimulatory DNA molecule comprising CpGs; proinflammatory
stimuli released from necrotic cells; urate crystals; activated
component of the complement cascade; activated component of immune
complexes; complement receptor agonist; cytokine; or cytokine
receptor agonist.
7. The method of claim 6, wherein the adjuvant comprises an agonist
of Toll-Like Receptor 2, 3, 4, 7, 8 or 9.
8. The method of any one of claims 1-3, wherein the single adjuvant
is coupled to the synthetic nanocarriers of the population of
synthetic nanocarriers.
9. The method of any one of claims 1-3, wherein the adjuvant is
coupled to another population of synthetic nanocarriers, and the
other population of synthetic nanocarriers is administered to the
subject.
10. The method of any one of claims 1-3, wherein the adjuvant is
not coupled to a synthetic nanocarrier.
11. The method of any one of claims 1-3, wherein the synthetic
nanocarriers comprise a polymeric nanoparticle, a metallic
nanoparticle, a dendrimer, a buckyball, a nanowire, a virus-like
particle or a peptide or protein particle.
12. The method of any one of claims 1-3, wherein the first protein
and/or one or more additional antigens are antigens associated with
cancer, an infection or infectious disease, a non-autoimmune or
degenerative disease, HIV, malaria, leischmania, human filovirus,
togavirus, alphavirus, arenavirus, bunyavirus, flavivirus, human
papillomavirus, human influenza A virus, hepatitis B or hepatitis
C.
13. The method of any one of claims 1-3, wherein the subject has or
is at risk of having cancer, an infection or infectious disease or
a non-autoimmune or degenerative disease.
14. The method of any one of claims 1-3, wherein, when the
synthetic nanocarriers have a minimum dimension of equal to or less
than 100 nm, the synthetic nanocarriers do not comprise a surface
with hydroxyl groups that substantially activate complement.
15. The method of any one of claims 1-3, wherein the synthetic
nanocarriers comprise one or more polymers.
16. The method of claim 15, wherein the one or more polymers
comprise a polyester, polyamino acid, polycarbonate, polyacetal,
polyketal, polysaccharide, polyethyloxazoline or
polyethyleneimine.
17. The method of claim 16, wherein the one or more polymers
comprise a polyester which comprises a poly(lactic acid),
poly(glycolic acid), poly(lactic-co-glycolic acid) or
polycaprolactone.
18. The method of claim 17, wherein the one or more polymers is
coupled to a polyether.
19. The method of claim 18, wherein the polyether comprises
polyethylene glycol.
Description
FIELD OF THE INVENTION
This invention relates to methods for generating humoral and
cytotoxic T lymphocyte (CTL) immune responses in a subject and
related compositions. Generally, the humoral and CTL immune
responses are generated with synthetic nanocarrier compositions
that comprise a protein that comprises at least one humoral epitope
and at least one MHC Class I-restricted epitope.
BACKGROUND OF THE INVENTION
Classically, vaccines have promoted a single arm of the immune
system, for example, the generation of a humoral immune response
consisting of antibodies to an antigen or, alternatively,
activation of a CTL response to an antigen. Additionally,
conventional vaccines generally do not target the sites of action
of cells of interest, such as APCs, in an optimal manner. Methods
and compositions for effectively activating both of these arms of
the immune system optimally to effectively generate immune
responses and/or reduce off-target effects and toxicity are
needed.
SUMMARY OF THE INVENTION
Provided herein are methods, and related compositions, for
generating humoral and CTL immune responses in a subject. In one
aspect, a method comprising identifying a subject in need of a
humoral and CTL immune response to a first protein, and
administering to the subject a composition comprising a population
of synthetic nanocarriers coupled to the first protein, wherein the
first protein comprises at least one humoral epitope and at least
one MHC Class I-restricted epitope that are not the same epitope,
wherein the population of synthetic nanocarriers does not comprise
a saponin-cholesterol adjuvant, and wherein the mean of a particle
size distribution obtained using dynamic light scattering of the
population of synthetic nanocarriers is a maximum dimension of from
20 nm to 250 nm is provided. In another embodiment, the mean of a
DLS distribution is determined by any of the examples of such a
method provided herein. Such examples are described in more detail
below. In one embodiment, the composition is administered in an
amount effective to generate a humoral and CTL immune response to
the first protein.
In another aspect, a method comprising administering to the subject
a composition comprising a population of synthetic nanocarriers
coupled to a first protein; wherein the first protein comprises at
least one humoral epitope and at least one MHC Class I-restricted
epitope that are not the same epitope, wherein the population of
synthetic nanocarriers does not comprise a saponin-cholesterol
adjuvant, wherein the mean of a particle size distribution obtained
using dynamic light scattering of the population of synthetic
nanocarriers is a maximum dimension of from 20 nm to 250 nm, and
wherein the composition is administered according to a vaccination
regimen is provided.
In another aspect, a method comprising administering to the subject
a composition comprising a population of synthetic nanocarriers
coupled to a first protein; wherein the first protein comprises at
least one humoral epitope and at least one MHC Class I-restricted
epitope that are not the same epitope, wherein the population of
synthetic nanocarriers does not comprise a saponin-cholesterol
adjuvant, wherein the mean of a particle size distribution obtained
using dynamic light scattering of the population of synthetic
nanocarriers is a maximum dimension of from 20 nm to 250 nm, and
wherein the composition is administered according to a protocol
that was previously shown to result in a humoral and CTL immune
response specific to the first protein in one or more test subjects
is provided.
In one embodiment, the methods provided herein further comprise
identifying a subject in need of a humoral and cytotoxic T
lymphocyte (CTL) immune response to the first protein. In another
embodiment, the composition is administered according to a
vaccination regimen. In yet another embodiment, the composition is
administered according to a protocol that was previously shown to
result in a humoral and CTL immune response specific to the first
protein in one or more test subjects
In another embodiment, the composition further comprises one or
more adjuvants. In another embodiment, the method further comprises
administering one or more adjuvants. In a further embodiment, the
one or more adjuvants comprise stimulators or agonists of pattern
recognition receptors, mineral salts, alum, alum combined with
monphosphoryl lipid A of Enterobacteria (MPL), MPL.RTM. (AS04),
AS15, saponins, QS-21, Quil-A, ISCOMs, ISCOMATRIX.TM., MF59.TM.,
Montanide.RTM. ISA 51, Montanide.RTM. ISA 720, AS02, liposomes and
liposomal formulations, AS01, synthesized or specifically prepared
microparticles and microcarriers, bacteria-derived outer membrane
vesicles of N. gonorrheae or Chlamydia trachomatis, chitosan
particles, depot-forming agents, Pluronic.RTM. block co-polymers,
specifically modified or prepared peptides, muramyl dipeptide,
aminoalkyl glucosaminide 4-phosphates, RC529, bacterial toxoids,
toxin fragments, agonists of Toll-Like Receptors 2, 3, 4, 5, 7, 8,
9 and/or combinations thereof; adenine derivatives;
immunostimulatory DNA; immunostimulatory RNA; imidazoquinoline
amines, imidazopyridine amines, 6,7-fused cycloalkylimidazopyridine
amines, 1,2-bridged imidazoquinoline amines; imiquimod; resiquimod;
agonist for DC surface molecule CD40; type I interferons; poly I:C;
bacterial lipopolysacccharide (LPS); VSV-G; HMGB-1; flagellin or
portions or derivatives thereof; immunostimulatory DNA molecules
comprising CpGs; proinflammatory stimuli released from necrotic
cells; urate crystals; activated components of the complement
cascade; activated components of immune complexes; complement
receptor agonists; cytokines; or cytokine receptor agonists. In yet
another embodiment, the one or more adjuvants comprise an agonist
of Toll-Like Receptor 2, 3, 4, 7, 8 or 9. In still another
embodiment, the one or more adjuvants comprise an imidazoquinoline
or oxoadenine. In one embodiment, the imidazoquinoline comprises
resiquimod or imiquimod.
In another embodiment, the one or more adjuvants are coupled to the
synthetic nanocarriers of the population of synthetic
nanocarriers.
In still another embodiment, the composition further comprises or
the method further comprises administering another population of
synthetic nanocarriers, and the one or more adjuvants are coupled
to the synthetic nanocarriers of the other population of synthetic
nanocarriers.
In a further embodiment, the one or more adjuvants are not coupled
to a synthetic nanocarrier.
In another embodiment, the composition further comprises one or
more additional antigens or the method further comprises
administering one or more additional antigens. In an embodiment,
the one or more additional antigens comprise at least one humoral
epitope and/or at least one MHC Class I-restricted epitope. In
another embodiment, the one or more additional antigens comprise at
least one humoral epitope and at least one MHC Class I-restricted
epitope that are not the same epitope. In one embodiment, the one
or more additional antigens comprise a second protein. In another
embodiment, the one or more additional antigens comprise a humoral
epitope and/or a MHC Class I-restricted epitope. In still another
embodiment, the one or more additional antigens comprise a humoral
epitope and a MHC Class I-restricted epitope. In yet another
embodiment, the one or more additional antigens comprise at least
one humoral epitope and at least one MHC Class I-restricted epitope
that are not the same epitope.
In one embodiment, the one or more additional antigens are coupled
to the synthetic nanocarriers.
In another embodiment, the composition further comprises or the
method further comprises administering another population of
synthetic nanocarriers, and the one or more additional antigens are
coupled to the synthetic nanocarriers of the other population of
synthetic nanocarriers.
In still another embodiment, the one or more additional antigens
are not coupled to a synthetic nanocarrier.
In one embodiment, the synthetic nanocarriers and/or other
synthetic nanocarriers comprise a polymeric nanoparticle, a
metallic nanoparticle, a dendrimer, a buckyball, a nanowire, a
virus-like particle or a peptide or protein particle. In another
embodiment, the synthetic nanocarriers and/or other synthetic
nanocarriers comprise one or more polymers. In yet another
embodiment, the one or more polymers comprise a polyester,
polyamino acid, polycarbonate, polyacetal, polyketal,
polysaccharide, polyethyloxazoline or polyethyleneimine. In still
another embodiment, the one or more polymers comprise a polyester.
In one embodiment, the polyester comprises a poly(lactic acid),
poly(glycolic acid), poly(lactic-co-glycolic acid) or
polycaprolactone. In another embodiment, the polyester is coupled
to a polyether. In yet another embodiment, the polyether comprises
polyethylene glycol.
In one embodiment, the first protein and/or one or more additional
antigens are antigens associated with cancer, an infection or
infectious disease or a non-autoimmune or degenerative disease. In
another embodiment, the first protein and/or one or more additional
antigens are antigens associated with human immunodeficiency virus
(HIV), malaria, leischmaniasis, a human filovirus infection, a
togavirus infection, a alphavirus infection, an arenavirus
infection, a bunyavirus infection, a flavivirus infection, a human
papillomavirus infection, a human influenza A virus infection, a
hepatitis B infection or a hepatitis C infection.
In another embodiment, the subject has or is at risk of having
cancer, an infection or infectious disease or a non-autoimmune or
degenerative disease. In yet another embodiment, the subject has or
is at risk of having HIV, malaria, leischmaniasis, a human
filovirus infection, a togavirus infection, a alphavirus infection,
an arenavirus infection, a bunyavirus infection, a flavivirus
infection, a human papillomavirus infection, a human influenza A
virus infection, a hepatitis B infection or a hepatitis C
infection.
In still another embodiment, the humoral and CTL immune responses
that are generated are clinically effective. In one embodiment, the
immune responses are effective to treat or prevent cancer, an
infection or infectious disease or a non-autoimmune or degenerative
disease in the subject. In another embodiment, the immune responses
are effective to treat or prevent HIV, malaria, leischmaniasis, a
human filovirus infection, a togavirus infection, a alphavirus
infection, an arenavirus infection, a bunyavirus infection, a
flavivirus infection, a human papillomavirus infection, a human
influenza A virus infection, a hepatitis B infection or a hepatitis
C infection in the subject.
In one embodiment, the composition further comprises a
pharmaceutically acceptable excipient.
In another embodiment, the composition is sterile.
In another embodiment, the composition is reconstituted from a
lyophilized form.
In another aspect, a dosage form comprising any of the compositions
provided is provided.
In yet another aspect, a vaccine comprising any of the compositions
and dosage forms provided is provided.
In yet a further embodiment, the composition is administered by
intravenous, oral, subcutaneous, pulmonary, intranasal,
intradermal, transmucosal, intramucosal or intramuscular
administration.
In still another aspect, a method comprising administering any of
the compositions provided herein to a subject in need thereof is
provided. In one embodiment, the subject is a human. In another
embodiment, the subject has or is at risk of having cancer. In
still another embodiment, the subject has or is at risk of having
an infection or infectious disease. In yet another embodiment, the
subject has or is at risk of having a non-autoimmune or
degenerative disease. In yet a further embodiment, the subject has
or is at risk of having HIV. In another embodiment, the subject has
or is at risk of having malaria, leischmaniasis, a human filovirus
infection, a togavirus infection, a alphavirus infection, an
arenavirus infection, a bunyavirus infection, a flavivirus
infection, a human papillomavirus infection, a human influenza A
virus infection, a hepatitis B infection or a hepatitis C
infection.
In one embodiment of any of the methods provided herein, any of the
compositions provided can be administered to a subject, such as a
human, according to a vaccination regimen.
In another embodiment of any of the method provided herein, any of
the compositions provided can be administered to a subject
according to a protocol that was previously shown to result in a
humoral and CTL immune response specific to the first protein in
one or more test subjects is provided
In another aspect, any of the methods provided can further comprise
assessing the humoral and CTL immune response in the subject. The
methods for assessing the humoral and CTL immune response can be
any of the methods provided herein.
In yet another aspect, a method comprising preparing any of the
compositions provided herein and assessing the generation of a
humoral and CTL immune response is provided. In one embodiment, the
composition comprises synthetic nanocarriers coupled to a first
protein that comprises at least one humoral epitope and at least
one MHC Class I-restricted epitope that are not the same epitope.
In another embodiment, the population of synthetic nanocarriers
coupled to the first protein does not comprise a
saponin-cholesterol adjuvant and/or the mean of a particle size
distribution obtained using dynamic light scattering of the
population of synthetic nanocarriers is a maximum dimension of from
20 nm to 250 nm.
In one aspect, a composition comprising a population of synthetic
nanocarriers coupled to a first protein, wherein the first protein
comprises at least one humoral epitope and at least one MHC Class
I-restricted epitope that are not the same epitope, wherein the
population of synthetic nanocarriers does not comprise a
saponin-cholesterol adjuvant, and wherein the mean of a particle
size distribution obtained using dynamic light scattering of the
population of synthetic nanocarriers is a maximum dimension of from
20 nm to 250 nm, for use in therapy or prophylaxis is provided.
In another aspect, a composition comprising a population of
synthetic nanocarriers coupled to a first protein, wherein the
first protein comprises at least one humoral epitope and at least
one MHC Class I-restricted epitope that are not the same epitope,
wherein the population of synthetic nanocarriers does not comprise
a saponin-cholesterol adjuvant, and wherein the mean of a particle
size distribution obtained using dynamic light scattering of the
population of synthetic nanocarriers is a maximum dimension of from
20 nm to 250 nm, for use in any of the methods provided herein is
provided.
In yet another aspect, a composition comprising a population of
synthetic nanocarriers coupled to a first protein, wherein the
first protein comprises at least one humoral epitope and at least
one MHC Class I-restricted epitope that are not the same epitope,
wherein the population of synthetic nanocarriers does not comprise
a saponin-cholesterol adjuvant, and wherein the mean of a particle
size distribution obtained using dynamic light scattering of the
population of synthetic nanocarriers is a maximum dimension of from
20 nm to 250 nm, for use in vaccination is provided.
In still another aspect, a composition comprising a population of
synthetic nanocarriers coupled to a first protein, wherein the
first protein comprises at least one humoral epitope and at least
one MHC Class I-restricted epitope that are not the same epitope,
wherein the population of synthetic nanocarriers does not comprise
a saponin-cholesterol adjuvant, and wherein the mean of a particle
size distribution obtained using dynamic light scattering of the
population of synthetic nanocarriers is a maximum dimension of from
20 nm to 250 nm, for use in generating a humoral and CTL immune
response to the first protein in a subject is provided. In one
embodiment, these immune responses are clinically effective. In
another embodiment, these immune responses are each effective in
achieving immunity against a disease.
In a further aspect, a composition comprising a population of
synthetic nanocarriers coupled to a first protein, wherein the
first protein comprises at least one humoral epitope and at least
one MHC Class I-restricted epitope that are not the same epitope,
wherein the population of synthetic nanocarriers does not comprise
a saponin-cholesterol adjuvant, and wherein the mean of a particle
size distribution obtained using dynamic light scattering of the
population of synthetic nanocarriers is a maximum dimension of from
20 nm to 250 nm, for use in a method of therapy or prophylaxis of
cancer, an infection or infectious disease, a non-autoimmune or
degenerative disease, HIV, malaria, leischmaniasis, a human
filovirus infection, a togavirus infection, a alphavirus infection,
an arenavirus infection, a bunyavirus infection, a flavivirus
infection, a human papillomavirus infection, a human influenza A
virus infection, a hepatitis B infection or a hepatitis C infection
I is provided.
In still a further aspect, a composition comprising a population of
synthetic nanocarriers coupled to a first protein, wherein the
first protein comprises at least one humoral epitope and at least
one MHC Class I-restricted epitope that are not the same epitope,
wherein the population of synthetic nanocarriers does not comprise
a saponin-cholesterol adjuvant, and wherein the mean of a particle
size distribution obtained using dynamic light scattering of the
population of synthetic nanocarriers is a maximum dimension of from
20 nm to 250 nm, for use in a method of therapy or prophylaxis
comprising administration by intravenous, oral, subcutaneous,
pulmonary, intranasal, intradermal, transmucosal, intramucosal or
intramuscular administration is provided.
In yet a further aspect, a composition comprising a population of
synthetic nanocarriers coupled to a first protein, wherein the
first protein comprises at least one humoral epitope and at least
one MHC Class I-restricted epitope that are not the same epitope,
wherein the population of synthetic nanocarriers does not comprise
a saponin-cholesterol adjuvant, and wherein the mean of a particle
size distribution obtained using dynamic light scattering of the
population of synthetic nanocarriers is a maximum dimension of from
20 nm to 250 nm, for the manufacture of a medicament, for example a
vaccine, for use in any of the methods provided herein is
provided.
In another aspect, a composition for use as defined for any of the
compositions or methods provided herein wherein the composition is
any of the compositions provided herein is provided.
BRIEF DESCRIPTION OF FIGURES
FIG. 1 shows the antibody titers generated at days 12, 26, 43 and
57 following a prime and one boost (on day 28) vaccination
regimen.
FIG. 2 shows the antibody titers generated at days 26 and 34
following a prime and one boost (on day 14) vaccination
regimen.
FIG. 3 shows the induction of local memory CTL response by
synthetic nanocarriers coupled to ovalbumin (OVA).
FIG. 4 shows the induction of central CTL response by synthetic
nanocarriers coupled to ovalbumin.
FIG. 5 shows the induction of central CTL induction by synthetic
nanocarrier compositions with ovalbumin and adjuvant.
FIG. 6 shows the antibody titers generated at days 25 and 42
following a prime and two boosts (on days 14 and 28) vaccination
regimen.
FIG. 7 shows the development of antibody titers after a single
injection by NC-R848+NC-OVA. Individual titers and averages for
each experimental group are shown. Nanocarrier=NC=NP.
FIG. 8 shows the specific cytotoxicity in vivo after a single
immunization with NC-R848+NC-OVA. Averages for each group with
standard deviation are shown.
FIG. 9 shows the anti-ovalbumin antibody titers upon the
immunization with nanocarriers carrying CpG and OVA vs. free CpG
(5.times.) and OVA (5.times.).
FIG. 10 shows the induction of OVA-specific CTL response in
draining lymph nodes and spleens by the immunization with
nanocarriers carrying CpG and OVA vs. free CpG (5.times.) and OVA
(5.times.).
FIG. 11 shows the expansion of systemically induced OVA-specific
CTLs in vitro upon the immunization with nanocarriers carrying CpG
and OVA vs. free CpG (5.times.) and OVA (5.times.). Left Y axis
(dark striped bars)-fraction of SIINFEKL (SEQ ID NO: 1)-specific
CD8+ cells after expansion; right Y axis (light striped
bars)-expansion potential presented as proportion of post- and
pre-expansion SIINFEKL (SEQ ID NO: 1)-specific CTLs.
DETAILED DESCRIPTION OF THE INVENTION
Before describing the present invention in detail, it is to be
understood that this invention is not limited to particularly
exemplified materials or process parameters as such may, of course,
vary. It is also to be understood that the terminology used herein
is for the purpose of describing particular embodiments of the
invention only, and is not intended to be limiting of the use of
alternative terminology to describe the present invention.
All publications, patents and patent applications cited herein,
whether supra or infra, are hereby incorporated by reference in
their entirety for all purposes.
As used in this specification and the appended claims, the singular
forms "a," "an" and "the" include plural referents unless the
content clearly dictates otherwise. For example, reference to "a
polymer" includes a mixture of two or more such molecules or a
mixture of differing molecular weights of a single polymer species,
reference to "a synthetic nanocarrier" includes a mixture of two or
more such synthetic nanocarriers or a plurality of such synthetic
nanocarriers, reference to "a DNA molecule" includes a mixture of
two or more such DNA molecules or a plurality of such DNA
molecules, reference to "an adjuvant" includes mixture of two or
more such adjuvant molecules or a plurality of such adjuvant
molecules, and the like.
As used herein, the term "comprise" or variations thereof such as
"comprises" or "comprising" are to be read to indicate the
inclusion of any recited integer (e.g. a feature, element,
characteristic, property, method/process step or limitation) or
group of integers (e.g. features, elements, characteristics,
properties, method/process steps or limitations) but not the
exclusion of any other integer or group of integers. Thus, as used
herein, the term "comprising" is inclusive and does not exclude
additional, unrecited integers or method/process steps.
In embodiments of any of the compositions and methods provided
herein, "comprising" may be replaced with "consisting essentially
of" or "consisting of". The phrase "consisting essentially of" is
used herein to require the specified integer(s) or steps as well as
those which do not materially affect the character or function of
the claimed invention. As used herein, the term "consisting" is
used to indicate the presence of the recited integer (e.g. a
feature, element, characteristic, property, method/process step or
limitation) or group of integers (e.g. features, elements,
characteristics, properties, method/process steps or limitations)
alone.
A. Introduction
Treatment of challenging diseases such as of HIV, malaria,
hepatitis B, and cancer with therapeutic or prophylactic vaccines
may be enhanced by, or in some circumstances require, combined
humoral and CTL immune responses. While vaccine approaches have
been proposed for creating a combined CTL and humoral immune
response, alternative approaches could provide valuable
improvements in clinical efficacy, safety, and/or
manufacturability. Provided herein are methods for using synthetic
nanocarrier compositions, and related compositions, that are
believed to have not been previously shown to generate strong and
effective humoral and CTL immune responses. Such compositions can
effectively target immune cells of interest to generate more
effective immune responses.
The inventors have unexpectedly and surprisingly discovered that
the problems and limitations noted above can be overcome by
practicing the invention disclosed herein. In particular, it has
been unexpectedly and surprisingly discovered that effective
humoral and CTL immune responses can be generated with synthetic
nanocarriers to which a protein, that comprises at least one
humoral epitope and at least one MHC Class I-restricted epitope
that are not the same epitope, is coupled, wherein the population
of synthetic nanocarriers does not comprise a saponin-cholesterol
adjuvant, and wherein the mean of a particle size distribution
obtained using dynamic light scattering of the population of
synthetic nanocarriers is a maximum dimension of from 20 nm to 250
nm. In one aspect, therefore, a method comprising administering
such a composition is provided. In one embodiment, the method
comprises identifying a subject in need of a humoral and CTL immune
response to a first protein, and administering to the subject a
composition comprising such synthetic nanocarriers. In some
embodiments, the composition is in an amount effective to generate
a humoral and CTL immune response to the first protein. In another
embodiment, the method comprises administering such a composition
to a subject according to a protocol that was previously shown to
result in a humoral and CTL immune response specific to the first
protein in one or more test subjects.
In embodiments, the immune responses that are generated are
clinically effective. In some embodiments, the subject to which the
compositions are administered may have or be at risk of having
cancer, an infection or infectious disease or a non-autoimmune or
degenerative disease. In other embodiments, the subject may have or
be at risk of having HIV, malaria, leischmaniasis, a human
filovirus infection, a togavirus infection, a alphavirus infection,
an arenavirus infection, a bunyavirus infection, a flavivirus
infection, a human papillomavirus infection, a human influenza A
virus infection, a hepatitis B infection or a hepatitis C
infection.
In other embodiments, the compositions are administered to a
subject, such as a human, according to a vaccination regimen.
The invention will now be described in more detail below.
B. Definitions
"Adjuvant" means an agent that does not constitute a specific
antigen, but boosts the strength and longevity of an immune
response to a concomitantly administered antigen. Such adjuvants
may include, but are not limited to stimulators of pattern
recognition receptors, such as Toll-like receptors, RIG-1 and
NOD-like receptors (NLR), mineral salts, such as alum, alum
combined with monphosphoryl lipid (MPL) A of Enterobacteria, such
as Escherihia coli, Salmonella minnesota, Salmonella typhimurium,
or Shigella flexneri or specifically with MPL.RTM. (AS04), MPL A of
above-mentioned bacteria separately, saponins, such as QS-21,
Quil-A, ISCOMs, ISCOMATRIX.TM., emulsions such as MF59.TM.,
Montanide.RTM. ISA 51 and ISA 720, AS02 (QS21+squalene+MPL.RTM.),
liposomes and liposomal formulations such as AS01, synthesized or
specifically prepared microparticles and microcarriers such as
bacteria-derived outer membrane vesicles (OMV) of N. gonorrheae,
Chlamydia trachomatis and others, or chitosan particles,
depot-forming agents, such as Pluronic.RTM. block co-polymers,
specifically modified or prepared peptides, such as muramyl
dipeptide, aminoalkyl glucosaminide 4-phosphates, such as RC529, or
proteins, such as bacterial toxoids or toxin fragments.
"Saponin-cholesterol adjuvants" are saponin adjuvants that are
stabilized by admixture with cholesterol. Such adjuvants include,
for example, ISCOMs and ISCOMATRIX adjuvants. Preferably, in some
embodiments, the synthetic nanocarriers provided herein are not or
do not comprise such adjuvants. In other embodiments, the
compositions provided herein do not comprise such adjuvants.
In embodiments, adjuvants comprise agonists for pattern recognition
receptors (PRR), including, but not limited to Toll-Like Receptors
(TLRs), specifically TLRs 2, 3, 4, 5, 7, 8, 9 and/or combinations
thereof. In other embodiments, adjuvants comprise agonists for
Toll-Like Receptors 3, agonists for Toll-Like Receptors 7 and 8, or
agonists for Toll-Like Receptor 9; preferably the recited adjuvants
comprise imidazoquinolines; such as R848; adenine derivatives, such
as those disclosed in U.S. Pat. No. 6,329,381 (Sumitomo
Pharmaceutical Company), US Published Patent Application
2010/0075995 to Biggadike et al., or WO 2010/018132 to Campos et
al.; immunostimulatory DNA; or immunostimulatory RNA. In specific
embodiments, synthetic nanocarriers incorporate as adjuvants
compounds that are agonists for toll-like receptors (TLRs) 7 &
8 ("TLR 7/8 agonists"). Of utility are the TLR 7/8 agonist
compounds disclosed in U.S. Pat. No. 6,696,076 to Tomai et al.,
including but not limited to imidazoquinoline amines,
imidazopyridine amines, 6,7-fused cycloalkylimidazopyridine amines,
and 1,2-bridged imidazoquinoline amines. Preferred adjuvants
comprise imiquimod and resiquimod (also known as R848). In specific
embodiments, an adjuvant may be an agonist for the DC surface
molecule CD40. In certain embodiments, to stimulate immunity rather
than tolerance, a synthetic nanocarrier incorporates an adjuvant
that promotes DC maturation (needed for priming of naive T cells)
and the production of cytokines, such as type I interferons, which
promote antibody immune responses. In embodiments, adjuvants also
may comprise immunostimulatory RNA molecules, such as but not
limited to dsRNA, poly I:C or poly I:poly C12U (available as
Ampligen.RTM., both poly I:C and poly I:polyC12U being known as
TLR3 stimulants), and/or those disclosed in F. Heil et al.,
"Species-Specific Recognition of Single-Stranded RNA via Toll-like
Receptor 7 and 8" Science 303(5663), 1526-1529 (2004); J. Vollmer
et al., "Immune modulation by chemically modified ribonucleosides
and oligoribonucleotides" WO 2008033432 A2; A. Forsbach et al.,
"Immunostimulatory oligoribonucleotides containing specific
sequence motif(s) and targeting the Toll-like receptor 8 pathway"
WO 2007062107 A2; E. Uhlmann et al., "Modified oligoribonucleotide
analogs with enhanced immunostimulatory activity" U.S. Pat. Appl.
Publ. US 2006241076; G. Lipford et al., "Immunostimulatory viral
RNA oligonucleotides and use for treating cancer and infections" WO
2005097993 A2; G. Lipford et al., "Immunostimulatory G,U-containing
oligoribonucleotides, compositions, and screening methods" WO
2003086280 A2. In some embodiments, an adjuvant may be a TLR-4
agonist, such as bacterial lipopolysacccharide (LPS), VSV-G, and/or
HMGB-1. In some embodiments, adjuvants may comprise TLR-5 agonists,
such as flagellin, or portions or derivatives thereof, including
but not limited to those disclosed in U.S. Pat. Nos. 6,130,082,
6,585,980, and 7,192,725. In specific embodiments, synthetic
nanocarriers incorporate a ligand for Toll-like receptor (TLR)-9,
such as immunostimulatory DNA molecules comprising CpGs, which
induce type I interferon secretion, and stimulate T and B cell
activation leading to increased antibody production and cytotoxic T
cell responses (Krieg et al., CpG motifs in bacterial DNA trigger
direct B cell activation. Nature. 1995. 374:546-549; Chu et al. CpG
oligodeoxynucleotides act as adjuvants that switch on T helper 1
(Th1) immunity. J. Exp. Med. 1997. 186:1623-1631; Lipford et al.
CpG-containing synthetic oligonucleotides promote B and cytotoxic T
cell responses to protein antigen: a new class of vaccine
adjuvants. Eur. J. Immunol. 1997. 27:2340-2344; Roman et al.
Immunostimulatory DNA sequences function as T helper-1-promoting
adjuvants. Nat. Med. 1997. 3:849-854; Davis et al. CpG DNA is a
potent enhancer of specific immunity in mice immunized with
recombinant hepatitis B surface antigen. J. Immunol. 1998.
160:870-876; Lipford et al., Bacterial DNA as immune cell
activator. Trends Microbiol. 1998. 6:496-500; U.S. Pat. No.
6,207,646 to Krieg et al.; U.S. Pat. No. 7,223,398 to Tuck et al.;
U.S. Pat. No. 7,250,403 to Van Nest et al.; or U.S. Pat. No.
7,566,703 to Krieg et al.
In some embodiments, adjuvants may be proinflammatory stimuli
released from necrotic cells (e.g., urate crystals). In some
embodiments, adjuvants may be activated components of the
complement cascade (e.g., CD21, CD35, etc.). In some embodiments,
adjuvants may be activated components of immune complexes. The
adjuvants also include complement receptor agonists, such as a
molecule that binds to CD21 or CD35. In some embodiments, the
complement receptor agonist induces endogenous complement
opsonization of the synthetic nanocarrier. In some embodiments,
adjuvants are cytokines, which are small proteins or biological
factors (in the range of 5 kD-20 kD) that are released by cells and
have specific effects on cell-cell interaction, communication and
behavior of other cells. In some embodiments, the cytokine receptor
agonist is a small molecule, antibody, fusion protein, or
aptamer.
In embodiments, at least a portion of the dose of adjuvant may be
coupled to synthetic nanocarriers, preferably, all of the dose of
adjuvant is coupled to synthetic nanocarriers. In other
embodiments, at least a portion of the dose of the adjuvant is not
coupled to the synthetic nanocarriers. In embodiments, the dose of
adjuvant comprises two or more types of adjuvants. For instance,
and without limitation, adjuvants that act on different TLR
receptors may be combined. As an example, in an embodiment a TLR
7/8 agonist may be combined with a TLR 9 agonist. In another
embodiment, a TLR 7/8 agonist may be combined with a TLR 9 agonist.
In yet another embodiment, a TLR 9 agonist may be combined with a
TLR 9 agonist.
"Administering" or "administration" means providing a material,
such as a drug, to a subject in a manner that is pharmacologically
useful.
"Amount effective" is any amount of a composition provided herein
that produces one or more desired responses, such as one or more
desired immune responses. This amount can be for in vitro or in
vivo purposes. For in vivo purposes, the amount can be one that a
clinician would believe may have a clinical benefit for a subject
in need of a humoral immune response and a CTL immune response to a
single protein. An effective amount that a clinician would believe
may have a clinical benefit for such a subject is also referred to
herein as a "clinically effective amount". In embodiments, both the
humoral immune response and the CTL immune response that is
elicited by a composition provided herein results in a clinical
effect from each of these arms of the immune system. In other
embodiments, clinically effective amounts are effective amounts
that can be helpful in the treatment of a subject with a disease or
condition in which a humoral immune response and a CTL immune
response to a single protein would provide a benefit. Such subjects
include, in some embodiments, those that have or are at risk of
having cancer, an infection or infectious disease or a
non-autoimmune or degenerative disease. In other embodiments, such
subjects include those that have or are at risk of having HIV,
malaria, leischmaniasis, a human filovirus infection, a togavirus
infection, a alphavirus infection, an arenavirus infection, a
bunyavirus infection, a flavivirus infection, a human
papillomavirus infection, a human influenza A virus infection, a
hepatitis B infection or a hepatitis C infection.
Amounts effective include those that involve the production of a
humoral immune response against a humoral epitope and a CTL immune
response against a MHC Class I-restricted epitope administered in
one of the compositions provided herein. A subject's humoral and
CTL immune response can be monitored by routine methods. An amount
that is effective to produce the desired immune responses as
provided herein can also be an amount of a composition provided
herein that produces a desired therapeutic endpoint or a desired
therapeutic result. In one embodiment, the amount that is effective
is one that provides effective immunity against a disease or agent
that causes a disease as provided herein. In another embodiment,
the immunity persists in the subject for at least 6, 12, 18, 24,
36, 48, 60 or more months. In still another embodiment, the
immunity results or persists due to the administration of a
composition provided herein according to a vaccination regimen.
Amounts effective will depend, of course, on the particular subject
being treated; the severity of a condition, disease or disorder;
the individual patient parameters including age, physical
condition, size and weight; the duration of the treatment; the
nature of concurrent therapy (if any); the specific route of
administration and like factors within the knowledge and expertise
of the health practitioner. These factors are well known to those
of ordinary skill in the art and can be addressed with no more than
routine experimentation. It is generally preferred that a maximum
dose be used, that is, the highest safe dose according to sound
medical judgment. It will be understood by those of ordinary skill
in the art, however, that a patient may insist upon a lower dose or
tolerable dose for medical reasons, psychological reasons or for
virtually any other reason.
In general, doses of the compositions of the invention can range
from about 10 .mu.g/kg to about 100,000 .mu.g/kg. In some
embodiments, the doses can range from about 0.1 mg/kg to about 100
mg/kg. In still other embodiments, the doses can range from about
0.1 mg/kg to about 25 mg/kg, about 25 mg/kg to about 50 mg/kg,
about 50 mg/kg to about 75 mg/kg or about 75 mg/kg to about 100
mg/kg. Alternatively, the dose can be administered based on the
number of synthetic nanocarriers. For example, useful doses include
greater than 10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9 or 10.sup.10
synthetic nanocarriers per dose. Other examples of useful doses
include from about 1.times.10.sup.6 to about 1.times.10.sup.10,
about 1.times.10.sup.7 to about 1.times.10.sup.9 or about
1.times.10.sup.8 to about 1.times.10.sup.9 synthetic nanocarriers
per dose.
"Antigen" means any antigen that can generate one or more immune
responses. The antigen may be one that generates a humoral and/or
CTL immune response. Such antigens include, but are not limited to
proteins, peptides, small molecules, oligosaccharides, and
carbohydrates. In some embodiments, such an antigen comprises a
non-protein antigen (i.e., not a protein or peptide antigen). In
some embodiments, an antigen that generates a humoral immune
response comprises a carbohydrate associated with an infectious
agent. In some embodiments, an antigen that generates a humoral
immune response comprises a glycoprotein or glycopeptide associated
with an infectious agent. The infectious agent can be a bacterium,
virus, fungus, protozoan, or parasite. Antigens may be B cell or T
cell antigens.
The synthetic nanocarrier compositions for use in the inventive
methods provided herein are coupled to an antigen that is a protein
that comprises at least one humoral epitope and at least one MHC
Class I-restricted epitope that is not the same epitope. Such
compositions can, in some embodiments, comprise one or more
additional antigens that may also be so limited but not necessarily
so. The one or more additional antigens for use in the methods and
compositions provided herein can be any antigen, which include
antigens that comprise humoral epitopes and/or MHC Class
I-restricted epitopes. The one or more additional antigens may also
include MHC Class II-restricted epitopes. In other embodiments, the
one or more additional antigens may be any antigen that generates a
humoral immune response. In still other embodiments, the one or
more additional antigens may be any of the T cell antigens
described herein, including a CD-1 restricted antigen. In yet other
embodiments, the one or more additional antigens may be a protein,
peptide, small molecule, oligosaccharide and carbohydrate.
In embodiments, antigens, including a protein that comprises at
least one humoral epitope and at least one MHC Class I-restricted
epitope that are not the same epitope, are coupled to the synthetic
nanocarriers. In other embodiments, the antigens are not coupled to
the synthetic nanocarriers. In yet other embodiments, the antigens
are encapsulated in the synthetic nanocarriers. "Type(s) of
antigens" means molecules that share the same, or substantially the
same, antigenic characteristics.
"Antigens associated" with a disease, disorder or condition
provided herein are antigens that can generate an undesired immune
response against, as a result of, or in conjunction with the
disease, disorder or condition; the cause of the disease, disorder
or condition (or a symptom or effect thereof); and/or can generate
an undesired immune response that is a symptom, result or effect of
the disease, disorder or condition. In some embodiments, such as
with cancer, such antigens are expressed in or on diseased cells,
such as cancer or tumor cells, but not in or on normal or healthy
cells (or non-diseased cells). Such antigens can also comprise an
antigen that is expressed in or on diseased cells and on normal or
healthy cells (or non-diseased cells) but is expressed in or on
diseased cells at a greater level than on normal or healthy cells
(or non-diseased cells). Preferably, the use of an antigen
associated with a disease or condition provided herein will not
lead to a substantial or detrimental immune response against normal
or healthy cells or will lead to a beneficial immune response
against the disease or condition that outweighs any immune response
against normal or healthy cells (or non-diseased cells). The
antigens associated with a disease or condition provided herein, in
some embodiments, are proteins that are coupled to synthetic
nanocarriers that comprise humoral and/or MHC Class I-restricted
epitopes. In other embodiments, such proteins comprise a humoral
and a MHC Class I-restricted epitope. In still other embodiments,
such proteins comprise at least one humoral epitope and at least
one MHC Class I-restricted epitope that are not the same epitope.
Examples of antigens, including the foregoing proteins, are
provided elsewhere herein.
"At least a portion of the dose" means at least some part of the
dose, ranging up to including all of the dose.
An "at risk" subject is one in which a health practitioner believes
has a chance of having a disease or condition as provided
herein.
"B cell antigen" means any antigen that is recognized by or
triggers an immune response in a B cell (e.g., an antigen that is
specifically recognized by a B cell or a receptor thereon). In some
embodiments, an antigen that is a T cell antigen is also a B cell
antigen. In other embodiments, the T cell antigen is not also a B
cell antigen. B cell antigens include, but are not limited to
proteins, peptides, small molecules, oligosaccharides and
carbohydrates.
"Couple" or "Coupled" or "Couples" (and the like) means to
chemically associate one entity (for example a moiety) with
another. In some embodiments, the coupling is covalent, meaning
that the coupling occurs in the context of the presence of a
covalent bond between the two entities. In non-covalent
embodiments, the non-covalent coupling is mediated by non-covalent
interactions including but not limited to charge interactions,
affinity interactions, metal coordination, physical adsorption,
host-guest interactions, hydrophobic interactions, TT stacking
interactions, hydrogen bonding interactions, van der Waals
interactions, magnetic interactions, electrostatic interactions,
dipole-dipole interactions, and/or combinations thereof. In
embodiments, encapsulation is a form of coupling.
"Cytotoxic T lymphocyte (CTL) immune response" means any
stimulation, induction or proliferation of cytotoxic T cells,
preferably cytotoxic T cells that are specific to an epitope, such
as a MHC Class I-restricted epitope. In embodiments, the epitope is
or is of an antigen that is associated with any of the diseases or
conditions provided herein. Methods for assessing CTL immune
responses are known to those of skill in the art. Examples of such
a method are provided in the EXAMPLES.
"Dosage form" means a pharmacologically and/or immunologically
active material in a medium, carrier, vehicle, or device suitable
for administration to a subject.
"Encapsulate" means to enclose at least a portion of a substance
within a synthetic nanocarrier. In some embodiments, a substance is
enclosed completely within a synthetic nanocarrier. In other
embodiments, most or all of a substance that is encapsulated is not
exposed to the local environment external to the synthetic
nanocarrier. In other embodiments, no more than 50%, 40%, 30%, 20%,
10% or 5% (weight/weight) is exposed to the local environment.
Encapsulation is distinct from absorption, which places most or all
of a substance on a surface of a synthetic nanocarrier, and leaves
the substance exposed to the local environment external to the
synthetic nanocarrier.
"Epitope", also known as an antigenic determinant, is the part of
an antigen that is recognized by the immune system, specifically
by, for example, antibodies, B cells, or T cells. As used herein, a
"humoral epitope" is one that is recognized by antibodies or B
cells, while a "MHC Class I-restricted epitope" is one that is
presented to immune cells by MHC class I molecules found on
nucleated cells. "MHC Class II-restricted epitopes" are epitopes
that are presented to immune cells by MHC class II molecules found
on antigen presenting cells (APCs), for example, on professional
antigen-presenting immune cells, such as on macrophages, B cells,
and dendritic cells, or on non-hematopoietic cells, such as
hepatocytes.
A number of epitopes are known to those of skill in the art, and
exemplary epitopes suitable according to some aspects of this
invention include, but are not limited to those listed in the
Immune Epitope Database (www.immuneepitope.org, Vita R, Zarebski L,
Greenbaum J A, Emami H, Hoof I, Salimi N, Damle R, Sette A, Peters
B. The immune epitope database 2.0. Nucleic Acids Res. 2010
January; 38(Database issue):D854-62; the entire contents of which
as well as all database entries of IEDB version 2.4, August 2011,
and particularly all epitopes disclosed therein, are incorporated
herein by reference). Epitopes can also be identified with publicly
available algorithms, for example, the algorithms described in Wang
P, Sidney J, Kim Y, Sette A, Lund O, Nielsen M, Peters B. 2010.
peptide binding predictions for HLA DR, DP and DQ molecules. BMC
Bioinformatics 2010, 11:568; Wang P, Sidney J, Dow C, Mothe B,
Sette A, Peters B. 2008. A systematic assessment of MHC class II
peptide binding predictions and evaluation of a consensus approach.
PLoS Comput Biol. 4(4):e1000048; Nielsen M, Lund O. 2009.
N,N-align. An artificial neural network-based alignment algorithm
for MHC class II peptide binding prediction. BMC Bioinformatics.
10:296; Nielsen M, Lundegaard C, Lund O. 2007. Prediction of MHC
class II binding affinity using SMM-align, a novel stabilization
matrix alignment method. BMC Bioinformatics. 8:238; Bui H H, Sidney
J, Peters B, Sathiamurthy M, Sinichi A, Purton K A, Mothe B R,
Chisari F V, Watkins D I, Sette A. 2005. Immunogenetics.
57:304-314; Sturniolo T, Bono E, Ding J, Raddrizzani L, Tuereci O,
Sahin U, Braxenthaler M, Gallazzi F, Protti M P, Sinigaglia F,
Hammer J. 1999. Generation of tissue-specific and promiscuous HLA
ligand databases using DNA microarrays and virtual HLA class II
matrices. Nat. Biotechnol. 17(6):555-561; Nielsen M, Lundegaard C,
Worning P, Lauemoller S L, Lamberth K, Buus S, Brunak S, Lund O.
2003. Reliable prediction of T-cell epitopes using neural networks
with novel sequence representations. Protein Sci 12:1007-1017; Bui
H H, Sidney J, Peters B, Sathiamurthy M, Sinichi A, Purton K A,
Mothe B R, Chisari F V, Watkins D I, Sette A. 2005. Automated
generation and evaluation of specific MHC binding predictive tools:
ARB matrix applications. Immunogenetics 57:304-314; Peters B, Sette
A. 2005. Generating quantitative models describing the sequence
specificity of biological processes with the stabilized matrix
method. BMC Bioinformatics 6:132; Chou P Y, Fasman G D. 1978.
Prediction of the secondary structure of proteins from their amino
acid sequence. Adv Enzymol Relat Areas Mol Biol 47:45-148; Emini E
A, Hughes J V, Perlow D S, Boger J. 1985. Induction of hepatitis A
virus-neutralizing antibody by a virus-specific synthetic peptide.
J Virol 55:836-839; Karplus P A, Schulz G E. 1985. Prediction of
chain flexibility in proteins. Naturwissenschaften 72:212-213;
Kolaskar A S, Tongaonkar P C. 1990. A semi-empirical method for
prediction of antigenic determinants on protein antigens. FEBS Lett
276:172-174; Parker J M, Guo D, Hodges R S. 1986. New
hydrophilicity scale derived from high-performance liquid
chromatography peptide retention data: correlation of predicted
surface residues with antigenicity and X-ray-derived accessible
sites. Biochemistry 25:5425-5432; Larsen J E, Lund O, Nielsen M.
2006. Improved method for predicting linear B-cell epitopes.
Immunome Res 2:2; Ponomarenko J V, Bourne P E. 2007.
Antibody-protein interactions: benchmark datasets and prediction
tools evaluation. BMC Struct Biol 7:64; Haste Andersen P, Nielsen
M, Lund O. 2006. Prediction of residues in discontinuous B-cell
epitopes using protein 3D structures. Protein Sci 15:2558-2567;
Ponomarenko J V, Bui H, Li W, Fusseder N, Bourne P E, Sette A,
Peters B. 2008. ElliPro: a new structure-based tool for the
prediction of antibody epitopes. BMC Bioinformatics 9:514; Nielsen
M, Lundegaard C, Blicher T, Peters B, Sette A, Justesen S, Buus S,
and Lund 0.2008. PLoS Comput Biol. 4(7)e1000107. Quantitative
predictions of peptide binding to any HLA-DR molecule of known
sequence: NetMHCIIpan; the entire contents of each of which are
incorporated herein by reference for disclosure of methods and
algorithms for the identification of epitopes.
"Generating" means causing an action, such as an immune response
(e.g., a humoral immune response or a CTL immune response) against
an epitope to occur, either directly oneself or indirectly, such
as, but not limited to, an unrelated third party that takes an
action through reliance on one's words or deeds.
"Humoral immune response" means any immune response that results in
the production or stimulation of B cells and/or the production of
antibodies. Preferably, the humoral immune response is specific to
an epitope comprised within an inventive composition or
administered during the practice of an inventive method. Methods
for assessing whether a humoral response is induced are known to
those of ordinary skill in the art. The production of antibodies is
referred to herein as an "antibody response". "Antibody titer"
means the production of a measurable level of antibodies.
Preferably, the antibody response or generation of the antibody
titer is in a human. In some embodiments, the antibodies are
antibodies of a certain isotype, such as IgG or a subclass thereof.
Methods for measuring antibody titers are known in the art and
include Enzyme-linked Immunosorbent Assay (ELISA). Methods for
measuring antibody titers are also described in some detail in the
EXAMPLES. Preferably, the antibody response or antibody titer is
specific to an epitope as provided herein. In embodiments, the
antibody response can be quantitated, for example, as the number of
antibodies, concentration of antibodies or titer. The values can be
absolute or they can be relative. Assays for quantifying an
antibody response include antibody capture assays, enzyme-linked
immunosorbent assays (ELISAs), inhibition liquid phase absorption
assays (ILPAAs), rocket immunoelectrophoresis (RIE) assays and line
immunoelectrophoresis (LIE) assays. When an antibody response is
compared to another antibody response the same type of quantitative
value (e.g., titer) and method of measurement (e.g., ELISA) is
preferably used to make the comparison.
An ELISA method for measuring an antibody titer, for example, may
consist of the following steps (i) preparing an ELISA-plate coating
material such that the antibody target of interest is coupled to a
substrate polymer or other suitable material (ii) preparing the
coating material in an aqueous solution (such as PBS) and
delivering the coating material solution to the wells of a
multiwell plate for overnight deposition of the coating onto the
multiwell plate (iii) thoroughly washing the multiwell plate with
wash buffer (such as 0.05% Tween-20 in PBS) to remove excess
coating material (iv) blocking the plate for nonspecific binding by
applying a diluent solution (such as 10% fetal bovine serum in
PBS), (v) washing the blocking/diluent solution from the plate with
wash buffer (vi) diluting the serum sample(s) containing antibodies
and appropriate standards (positive controls) with diluent as
required to obtain a concentration that suitably saturates the
ELISA response (vii) serially diluting the plasma samples on the
multiwell plate such to cover a range of concentrations suitable
for generating an ELISA response curve (viii) incubating the plate
to provide for antibody-target binding (ix) washing the plate with
wash buffer to remove antibodies not bound to antigen (x) adding an
appropriate concentration of a secondary detection antibody in same
diluent such as a biotin-coupled detection antibody capable of
binding the primary antibody (xi) incubating the plate with the
applied detection antibody, followed by washing with wash buffer
(xii) adding an enzyme such as streptavidin-HRP (horse radish
peroxidase) that will bind to biotin found on biotinylated
antibodies and incubating (xiii) washing the multiwell plate (xiv)
adding substrate(s) (such as TMB solution) to the plate (xv)
applying a stop solution (such as 2N sulfuric acid) when color
development is complete (xvi) reading optical density of the plate
wells at a specific wavelength for the substrate (450 nm with
subtraction of readings at 570 nm) (xvi) applying a suitable
multiparameter curve fit to the data and defining half-maximal
effective concentration (EC50) as the concentration on the curve at
which half the maximum OD value for the plate standards is
achieved.
"Identifying" is any action or set of actions that allows a
clinician to recognize a subject as one who may benefit from the
methods and compositions provided herein. Preferably, the
identified subject is one who is in need of a humoral immune
response and CTL immune response to a single protein. Such subjects
include any subject that has or is at risk of having any of the
disease or conditions provided herein. The action or set of actions
may be either directly oneself or indirectly, such as, but not
limited to, an unrelated third party that takes an action through
reliance on one's words or deeds.
An "infection" or "infectious disease" is any condition or disease
caused by a microorganism, pathogen or other agent, such as a
bacterium, fungus, prion or virus.
"Maximum dimension of a synthetic nanocarrier" means the largest
dimension of a nanocarrier measured along any axis of the synthetic
nanocarrier. "Minimum dimension of a synthetic nanocarrier" means
the smallest dimension of a synthetic nanocarrier measured along
any axis of the synthetic nanocarrier. For example, for a
spheroidal synthetic nanocarrier, the maximum and minimum dimension
of a synthetic nanocarrier would be substantially identical, and
would be the size of its diameter. Similarly, for a cuboidal
synthetic nanocarrier, the minimum dimension of a synthetic
nanocarrier would be the smallest of its height, width or length,
while the maximum dimension of a synthetic nanocarrier would be the
largest of its height, width or length. In an embodiment, a minimum
dimension of at least 75%, preferably at least 80%, more preferably
at least 90%, of the synthetic nanocarriers in a sample, based on
the total number of synthetic nanocarriers in the sample, is equal
to or greater than 100 nm. In an embodiment, a maximum dimension of
at least 75%, preferably at least 80%, more preferably at least
90%, of the synthetic nanocarriers in a sample, based on the total
number of synthetic nanocarriers in the sample, is equal to or less
than 5 .mu.m. Preferably, a minimum dimension of at least 75%,
preferably at least 80%, more preferably at least 90%, of the
synthetic nanocarriers in a sample, based on the total number of
synthetic nanocarriers in the sample, is greater than 110 nm, more
preferably greater than 120 nm, more preferably greater than 130
nm, and more preferably still greater than 150 nm. Aspects ratios
of the maximum and minimum dimensions of inventive synthetic
nanocarriers may vary depending on the embodiment. For instance,
aspect ratios of the maximum to minimum dimensions of the synthetic
nanocarriers may vary from 1:1 to 1,000,000:1, preferably from 1:1
to 100,000:1, more preferably from 1:1 to 10,000:1, more preferably
from 1:1 to 1000:1, still more preferably from 1:1 to 100:1, and
yet more preferably from 1:1 to 10:1. Preferably, a maximum
dimension of at least 75%, preferably at least 80%, more preferably
at least 90%, of the synthetic nanocarriers in a sample, based on
the total number of synthetic nanocarriers in the sample is equal
to or less than 3 .mu.m, more preferably equal to or less than 2
.mu.m, more preferably equal to or less than 1 .mu.m, more
preferably equal to or less than 800 nm, more preferably equal to
or less than 600 nm, and more preferably still equal to or less
than 500 nm. In preferred embodiments, a minimum dimension of at
least 75%, preferably at least 80%, more preferably at least 90%,
of the synthetic nanocarriers in a sample, based on the total
number of synthetic nanocarriers in the sample, is equal to or
greater than 100 nm, more preferably equal to or greater than 120
nm, more preferably equal to or greater than 130 nm, more
preferably equal to or greater than 140 nm, and more preferably
still equal to or greater than 150 nm. Measurement of synthetic
nanocarrier dimensions (e.g., diameter) is obtained by suspending
the synthetic nanocarriers in a liquid (usually aqueous) media and
using dynamic light scattering (DLS) (e.g. using a Brookhaven
ZetaPALS instrument). For example, a suspension of synthetic
nanocarriers can be diluted from an aqueous buffer into purified
water to achieve a final synthetic nanocarrier suspension
concentration of approximately 0.01 to 0.1 mg/mL. The diluted
suspension may be prepared directly inside, or transferred to, a
suitable cuvette for DLS analysis. The cuvette may then be placed
in the DLS, allowed to equilibrate to the controlled temperature,
and then scanned for sufficient time to acquire a stable and
reproducible distribution based on appropriate inputs for viscosity
of the medium and refractive indicies of the sample. The effective
diameter, or mean of the distribution, is then reported.
"Dimension" or "size" or "diameter" of synthetic nanocarriers means
the mean of a particle size distribution obtained using dynamic
light scattering.
"MHC" refers to major histocompatibility complex, a large genomic
region or gene family found in most vertebrates that encodes MHC
molecules that display fragments or epitopes of processed proteins
on the cell surface. The presentation of MHC:peptide on cell
surfaces allows for surveillance by immune cells, usually a T cell.
There are two general classes of MHC molecules: Class I and Class
II. Generally, Class I MHC molecules are found on nucleated cells
and present peptides to cytotoxic T cells. Class II MHC molecules
are found on certain immune cells, chiefly macrophages, B cells and
dendritic cells, collectively known as APCs. The best-known genes
in the MHC region are the subset that encodes antigen-presenting
proteins on the cell surface. In humans, these genes are referred
to as human leukocyte antigen (HLA) genes.
"Pharmaceutically acceptable excipient" means a pharmacologically
inactive material used together with the recited synthetic
nanocarriers to formulate the compositions. Pharmaceutically
acceptable excipients comprise a variety of materials known in the
art, including but not limited to saccharides (such as glucose,
lactose, and the like), preservatives such as antimicrobial agents,
reconstitution aids, colorants, saline (such as phosphate buffered
saline), and buffers.
"Protein(s)" means compounds, typically having a molecular weight
greater than 1000 daltons, comprising amino acid residues joined
together primarily by peptide bonds between the carboxyl and amino
groups of adjacent amino acid residues. Proteins may also comprise
additional bonding structures such as secondary structures,
tertiary structures, and the like. Certain of the peptide bonds in
proteins may be replaced by other bond types, for various purposes,
such as stabilization or coupling. When coupled to synthetic
nanocarriers, preferably, there are multiple copies of the protein
that are coupled to each synthetic nanocarrier.
"Protocol" refers to any dosing regimen of one or more substances
to a subject. A dosing regimen may include the amount, frequency
and/or mode of administration. In some embodiments, such a protocol
may be used to administer one or more compositions of the invention
to one or more test subjects. Immune responses in these test
subjects can then be assessed to determine whether or not the
protocol was effective in generating desired immune response(s).
Any other therapeutic and/or prophylactic effects may also be
assessed instead of or in addition to the aforementioned immune
responses. Whether or not a protocol had a desired effect can be
determined using any of the methods provided herein or otherwise
known in the art. For example, a population of cells may be
obtained from a subject to which a composition provided herein has
been administered according to a specific protocol in order to
determine whether or not specific immune cells, cytokines,
antibodies, etc. were generated, activated, etc. Useful methods for
detecting the presence and/or number of immune cells include, but
are not limited to, flow cytometric methods (e.g., FACS) and
immunohistochemistry methods. Antibodies and other binding agents
for specific staining of immune cell markers, are commercially
available. Such kits typically include staining reagents for
multiple antigens that allow for FACS-based detection, separation
and/or quantitation of a desired cell population from a
heterogeneous population of cells.
"Subject" means animals, including warm blooded mammals such as
humans and primates; avians; domestic household or farm animals
such as cats, dogs, sheep, goats, cattle, horses and pigs;
laboratory animals such as mice, rats and guinea pigs; fish;
reptiles; zoo and wild animals; and the like.
"Synthetic nanocarrier(s)" means a discrete object that is not
found in nature, and that possesses at least one dimension that is
less than or equal to 5 microns in size. Albumin nanoparticles are
generally included as synthetic nanocarriers, however in certain
embodiments the synthetic nanocarriers do not comprise albumin
nanoparticles. In embodiments, synthetic nanocarriers do not
comprise chitosan. In certain other embodiments, the synthetic
nanocarriers do not comprise chitosan. In other embodiments,
inventive synthetic nanocarriers are not lipid-based nanoparticles.
In further embodiments, inventive synthetic nanocarriers do not
comprise a phospholipid.
A synthetic nanocarrier can be, but is not limited to, one or a
plurality of lipid-based nanoparticles (also referred to herein as
lipid nanoparticles, i.e., nanoparticles where the majority of the
material that makes up their structure are lipids), polymeric
nanoparticles, metallic nanoparticles, surfactant-based emulsions,
dendrimers, buckyballs, nanowires, virus-like particles (i.e.,
particles that are primarily made up of viral structural proteins
but that are not infectious or have low infectivity), peptide or
protein-based particles (also referred to herein as protein
particles, i.e., particles where the majority of the material that
makes up their structure are peptides or proteins) (such as albumin
nanoparticles) and/or nanoparticles that are developed using a
combination of nanomaterials such as lipid-polymer nanoparticles.
Synthetic nanocarriers may be a variety of different shapes,
including but not limited to spheroidal, cuboidal, pyramidal,
oblong, cylindrical, toroidal, and the like. Synthetic nanocarriers
according to the invention comprise one or more surfaces. Exemplary
synthetic nanocarriers that can be adapted for use in the practice
of the present invention comprise: (1) the biodegradable
nanoparticles disclosed in U.S. Pat. No. 5,543,158 to Gref et al.,
(2) the polymeric nanoparticles of Published US Patent Application
20060002852 to Saltzman et al., (3) the lithographically
constructed nanoparticles of Published US Patent Application
20090028910 to DeSimone et al., (4) the disclosure of WO
2009/051837 to von Andrian et al., (5) the nanoparticles disclosed
in Published US Patent Application 2008/0145441 to Penades et al.,
(6) the protein nanoparticles disclosed in Published US Patent
Application 20090226525 to de los Rios et al., (7) the virus-like
particles disclosed in published US Patent Application 20060222652
to Sebbel et al., (8) the nucleic acid coupled virus-like particles
disclosed in published US Patent Application 20060251677 to
Bachmann et al., (9) the virus-like particles disclosed in
WO2010047839A1 or WO2009106999A2, (10) the nanoprecipitated
nanoparticles disclosed in P. Paolicelli et al., "Surface-modified
PLGA-based Nanoparticles that can Efficiently Associate and Deliver
Virus-like Particles" Nanomedicine. 5(6):843-853 (2010) or (11)
apoptotic cells, apoptotic bodies or the synthetic or semisynthetic
mimics disclosed in U.S. Publication 2002/0086049. In embodiments,
synthetic nanocarriers may possess an aspect ratio greater than
1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7, or greater than 1:10.
Synthetic nanocarriers according to the invention that have a
minimum dimension of equal to or less than about 100 nm, preferably
equal to or less than 100 nm, do not comprise a surface with
hydroxyl groups that activate complement or alternatively comprise
a surface that consists essentially of moieties that are not
hydroxyl groups that activate complement. In a preferred
embodiment, synthetic nanocarriers according to the invention that
have a minimum dimension of equal to or less than about 100 nm,
preferably equal to or less than 100 nm, do not comprise a surface
that substantially activates complement or alternatively comprise a
surface that consists essentially of moieties that do not
substantially activate complement. In a more preferred embodiment,
synthetic nanocarriers according to the invention that have a
minimum dimension of equal to or less than about 100 nm, preferably
equal to or less than 100 nm, do not comprise a surface that
activates complement or alternatively comprise a surface that
consists essentially of moieties that do not activate complement.
In embodiments, synthetic nanocarriers exclude virus-like
particles. In embodiments, when synthetic nanocarriers comprise
virus-like particles, the virus-like particles comprise non-natural
adjuvant (meaning that the VLPs comprise an adjuvant other than
naturally occurring RNA generated during the production of the
VLPs). In embodiments, synthetic nanocarriers may possess an aspect
ratio greater than 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7, or
greater than 1:10.
"T cell antigen" means any antigen that is recognized by and
triggers an immune response in a T cell (e.g., an antigen that is
specifically recognized by a T cell receptor on a T cell or an NKT
cell via presentation of the antigen or portion thereof bound to a
Class I or Class II major histocompatability complex molecule
(MHC), or bound to a CD1 complex). In some embodiments, an antigen
that is a T cell antigen is also a B cell antigen. In other
embodiments, the T cell antigen is not also a B cell antigen. T
cell antigens generally are proteins or peptides. T cell antigens
may be an antigen that stimulates a CD8+ T cell response, a CD4+ T
cell response, or both. The nanocarriers, therefore, in some
embodiments can effectively stimulate both types of responses.
In some embodiments the T cell antigen is a T helper cell antigen
(i.e. one that can generate an enhanced response to a B cell
antigen, preferably an unrelated B cell antigen, through
stimulation of T cell help). In embodiments, a T helper cell
antigen may comprise one or more peptides obtained or derived from
tetanus toxoid, Epstein-Barr virus, influenza virus, respiratory
syncytial virus, measles virus, mumps virus, rubella virus,
cytomegalovirus, adenovirus, diphtheria toxoid, or a PADRE peptide
(known from the work of Sette et al. U.S. Pat. No. 7,202,351). In
other embodiments, a T helper cell antigen may comprise one or more
lipids, or glycolipids, including but not limited to:
.alpha.-galactosylceramide (.alpha.-GalCer), .alpha.-linked
glycosphingolipids (from Sphingomonas spp.), galactosyl
diacylglycerols (from Borrelia burgdorferi), lypophosphoglycan
(from Leishmania donovani), and phosphatidylinositol tetramannoside
(PIM4) (from Mycobacterium leprae). For additional lipids and/or
glycolipids useful as a T helper cell antigen, see V. Cerundolo et
al., "Harnessing invariant NKT cells in vaccination strategies."
Nature Rev Immun, 9:28-38 (2009). In embodiments, CD4+ T-cell
antigens may be derivatives of a CD4+ T-cell antigen that is
obtained from a source, such as a natural source. In such
embodiments, CD4+ T-cell antigen sequences, such as those peptides
that bind to MHC II, may have at least 70%, 80%, 90%, or 95%
identity to the antigen obtained from the source. In embodiments,
the T cell antigen, preferably a T helper cell antigen, may be
coupled to, or uncoupled from, a synthetic nanocarrier. In some
embodiments, the T cell antigen is encapsulated in the synthetic
nanocarriers of the compositions.
"Vaccine" means a composition of matter that improves the immune
response to a particular pathogen or disease. A vaccine typically
contains factors that stimulate a subject's immune system to
recognize a specific antigen as foreign and eliminate it from the
subject's body. A vaccine also establishes an immunologic `memory`
so the antigen will be quickly recognized and responded to if a
person is re-challenged. Vaccines can be prophylactic (for example
to prevent future infection by any pathogen), or therapeutic (for
example a vaccine against a tumor specific antigen for the
treatment of cancer). In embodiments, a vaccine may comprise dosage
forms according to the invention.
"Vaccine regimen" or "vaccination regimen" is a schedule of one or
more vaccinations that includes the number and timing of doses of a
vaccine. Generally, vaccination regimens are intended to achieve
immunity against the development of a disease or condition.
Preferably, the vaccine regimen is one that achieves immunity via
both the humoral and CTL arms of the immune system.
C. Compositions for Use in the Inventive Methods
Provided herein are methods and related compositions for effective
humoral and CTL immune response generation. It has been found that
synthetic nanocarriers to which a protein that comprises at least
one humoral epitope and at least one MHC Class I-restricted epitope
that are not the same epitope, is coupled, wherein the synthetic
nanocarriers do not comprise a saponin-cholesterol adjuvant, and
wherein the mean of a particle size distribution obtained using
dynamic light scattering of the population of synthetic
nanocarriers is a maximum dimension of from 20 nm to 250 nm can be
used to generate effective and strong humoral and CTL immune
responses. The compositions provided can be used for a variety of
desired clinical endpoints such as for vaccination.
A wide variety of synthetic nanocarriers can be used according to
the invention. In some embodiments, synthetic nanocarriers are
spheres or spheroids. In some embodiments, synthetic nanocarriers
are flat or plate-shaped. In some embodiments, synthetic
nanocarriers are cubes or cubic. In some embodiments, synthetic
nanocarriers are ovals or ellipses. In some embodiments, synthetic
nanocarriers are cylinders, cones, or pyramids.
In some embodiments, it is desirable to use a population of
synthetic nanocarriers that is relatively uniform in terms of size,
shape, and/or composition so that each synthetic nanocarrier has
similar properties. For example, at least 80%, at least 90%, or at
least 95% of the synthetic nanocarriers, based on the total number
of synthetic nanocarriers, may have a minimum dimension or maximum
dimension that falls within 5%, 10%, or 20% of the average diameter
or average dimension of the synthetic nanocarriers. In some
embodiments, a population of synthetic nanocarriers may be
heterogeneous with respect to size, shape, and/or composition.
Synthetic nanocarriers can be solid or hollow and can comprise one
or more layers. In some embodiments, each layer has a unique
composition and unique properties relative to the other layer(s).
To give but one example, synthetic nanocarriers may have a
core/shell structure, wherein the core is one layer (e.g. a
polymeric core) and the shell is a second layer (e.g. a lipid
bilayer or monolayer). Synthetic nanocarriers may comprise a
plurality of different layers.
In some embodiments, synthetic nanocarriers may comprise metal
particles, quantum dots, ceramic particles, etc. In some
embodiments, a non-polymeric synthetic nanocarrier is an aggregate
of non-polymeric components, such as an aggregate of metal atoms
(e.g., gold atoms).
In some embodiments, synthetic nanocarriers may optionally comprise
one or more lipids. In some embodiments, a synthetic nanocarrier
may comprise a liposome. In some embodiments, a synthetic
nanocarrier may comprise a lipid bilayer. In some embodiments, a
synthetic nanocarrier may comprise a lipid monolayer. In some
embodiments, a synthetic nanocarrier may comprise a micelle. In
some embodiments, a synthetic nanocarrier may comprise a core
comprising a polymeric matrix surrounded by a lipid layer (e.g.,
lipid bilayer, lipid monolayer, etc.). In some embodiments, a
synthetic nanocarrier may comprise a non-polymeric core (e.g.,
metal particle, quantum dot, ceramic particle, bone particle, viral
particle, proteins, nucleic acids, carbohydrates, etc.) surrounded
by a lipid layer (e.g., lipid bilayer, lipid monolayer, etc.).
In some embodiments, synthetic nanocarriers can comprise one or
more polymers. In some embodiments, such a polymer can be
surrounded by a coating layer (e.g., liposome, lipid monolayer,
micelle, etc.). In some embodiments, various elements (i.e.,
components) of the synthetic nanocarriers can be coupled with the
polymer.
In some embodiments, a component can be covalently associated with
a polymeric matrix. In some embodiments, covalent association is
mediated by a linker. In some embodiments, a component can be
noncovalently associated with a polymeric matrix. For example, in
some embodiments, a component can be encapsulated within,
surrounded by, and/or dispersed throughout a polymeric matrix.
Alternatively or additionally, a component can be associated with a
polymeric matrix by hydrophobic interactions, charge interactions,
van der Waals forces, etc.
A wide variety of polymers and methods for forming polymeric
matrices therefrom are known conventionally. In general, a
polymeric matrix comprises one or more polymers.
The synthetic nanocarriers provided herein may be polymeric
nanocarriers. Polymers may be natural or unnatural (synthetic)
polymers. Polymers may be homopolymers or copolymers comprising two
or more monomers. In terms of sequence, copolymers may be random,
block, or comprise a combination of random and block sequences.
Typically, polymers in accordance with the present invention are
organic polymers.
In some embodiments, the synthetic nanocarriers comprise one or
more polymers that comprise a polyester, polycarbonate, polyamide,
or polyether, or unit thereof. In other embodiments, the polymer
comprises poly(ethylene glycol) (PEG), poly(lactic acid),
poly(glycolic acid), poly(lactic-co-glycolic acid), or a
polycaprolactone, or unit thereof. In some embodiments, it is
preferred that the polymer is biodegradable. Therefore, in these
embodiments, it is preferred that if the polymer comprises a
polyether, such as poly(ethylene glycol) or unit thereof, the
polymer comprises a block-co-polymer of a polyether and a
biodegradable polymer such that the polymer is biodegradable. In
other embodiments, the polymer does not solely comprise a polyether
or unit thereof, such as poly(ethylene glycol) or unit thereof. The
one or more polymers may be comprised within a polymeric synthetic
nanocarrier or may be comprised in a number of other different
types of synthetic nanocarriers.
Examples of polymers suitable for use in the present invention also
include, but are not limited to polyethylenes, polycarbonates (e.g.
poly(1,3-dioxan-2one)), polyanhydrides (e.g. poly(sebacic
anhydride)), polypropylfumerates, polyamides (e.g.
polycaprolactam), polyacetals, polyethers, polyesters (e.g.,
polylactide, polyglycolide, polylactide-co-glycolide,
polycaprolactone, polyhydroxyacid (e.g.
poly(.beta.-hydroxyalkanoate))), poly(orthoesters),
polycyanoacrylates, polyvinyl alcohols, polyurethanes,
polyphosphazenes, polyacrylates, polymethacrylates, polyureas,
polystyrenes, and polyamines, polylysine, polylysine-PEG
copolymers, and poly(ethyleneimine), poly(ethylene imine)-PEG
copolymers.
In some embodiments, polymers in accordance with the present
invention include polymers which have been approved for use in
humans by the U.S. Food and Drug Administration (FDA) under 21
C.F.R. .sctn. 177.2600, including but not limited to polyesters
(e.g., polylactic acid, poly(lactic-co-glycolic acid),
polycaprolactone, polyvalerolactone, poly(1,3-dioxan-2one));
polyanhydrides (e.g., poly(sebacic anhydride)); polyethers (e.g.,
polyethylene glycol); polyurethanes; polymethacrylates;
polyacrylates; and polycyanoacrylates.
In some embodiments, polymers can be hydrophilic. For example,
polymers may comprise anionic groups (e.g., phosphate group,
sulphate group, carboxylate group); cationic groups (e.g.,
quaternary amine group); or polar groups (e.g., hydroxyl group,
thiol group, amine group). In some embodiments, a synthetic
nanocarrier comprising a hydrophilic polymeric matrix generates a
hydrophilic environment within the synthetic nanocarrier. In some
embodiments, polymers can be hydrophobic. In some embodiments, a
synthetic nanocarrier comprising a hydrophobic polymeric matrix
generates a hydrophobic environment within the synthetic
nanocarrier. Selection of the hydrophilicity or hydrophobicity of
the polymer may have an impact on the nature of materials that are
incorporated (e.g. coupled) within the synthetic nanocarrier.
In some embodiments, polymers may be modified with one or more
moieties and/or functional groups. A variety of moieties or
functional groups can be used in accordance with the present
invention. In some embodiments, polymers may be modified with
polyethylene glycol (PEG), with a carbohydrate, and/or with acyclic
polyacetals derived from polysaccharides (Papisov, 2001, ACS
Symposium Series, 786:301). Certain embodiments may be made using
the general teachings of U.S. Pat. No. 5,543,158 to Gref et al., or
WO publication WO2009/051837 by Von Andrian et al.
In some embodiments, polymers may be modified with a lipid or fatty
acid group. In some embodiments, a fatty acid group may be one or
more of butyric, caproic, caprylic, capric, lauric, myristic,
palmitic, stearic, arachidic, behenic, or lignoceric acid. In some
embodiments, a fatty acid group may be one or more of palmitoleic,
oleic, vaccenic, linoleic, alpha-linoleic, gamma-linoleic,
arachidonic, gadoleic, arachidonic, eicosapentaenoic,
docosahexaenoic, or erucic acid.
In some embodiments, polymers may be polyesters, including
copolymers comprising lactic acid and glycolic acid units, such as
poly(lactic acid-co-glycolic acid) and poly(lactide-co-glycolide),
collectively referred to herein as "PLGA"; and homopolymers
comprising glycolic acid units, referred to herein as "PGA," and
lactic acid units, such as poly-L-lactic acid, poly-D-lactic acid,
poly-D,L-lactic acid, poly-L-lactide, poly-D-lactide, and
poly-D,L-lactide, collectively referred to herein as "PLA." In some
embodiments, exemplary polyesters include, for example,
polyhydroxyacids; PEG copolymers and copolymers of lactide and
glycolide (e.g., PLA-PEG copolymers, PGA-PEG copolymers, PLGA-PEG
copolymers, and derivatives thereof. In some embodiments,
polyesters include, for example, poly(caprolactone),
poly(caprolactone)-PEG copolymers, poly(L-lactide-co-L-lysine),
poly(serine ester), poly(4-hydroxy-L-proline ester),
poly[.alpha.-(4-aminobutyl)-L-glycolic acid], and derivatives
thereof.
In some embodiments, a polymer may be PLGA. PLGA is a biocompatible
and biodegradable co-polymer of lactic acid and glycolic acid, and
various forms of PLGA are characterized by the ratio of lactic
acid:glycolic acid. Lactic acid can be L-lactic acid, D-lactic
acid, or D,L-lactic acid. The degradation rate of PLGA can be
adjusted by altering the lactic acid:glycolic acid ratio. In some
embodiments, PLGA to be used in accordance with the present
invention is characterized by a lactic acid:glycolic acid ratio of
approximately 85:15, approximately 75:25, approximately 60:40,
approximately 50:50, approximately 40:60, approximately 25:75, or
approximately 15:85.
In some embodiments, polymers may be one or more acrylic polymers.
In certain embodiments, acrylic polymers include, for example,
acrylic acid and methacrylic acid copolymers, methyl methacrylate
copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate,
aminoalkyl methacrylate copolymer, poly(acrylic acid),
poly(methacrylic acid), methacrylic acid alkylamide copolymer,
poly(methyl methacrylate), poly(methacrylic acid anhydride), methyl
methacrylate, polymethacrylate, poly(methyl methacrylate)
copolymer, polyacrylamide, aminoalkyl methacrylate copolymer,
glycidyl methacrylate copolymers, polycyanoacrylates, and
combinations comprising one or more of the foregoing polymers.
The acrylic polymer may comprise fully-polymerized copolymers of
acrylic and methacrylic acid esters with a low content of
quaternary ammonium groups.
In some embodiments, polymers can be cationic polymers. In general,
cationic polymers are able to condense and/or protect negatively
charged strands of nucleic acids (e.g. DNA, or derivatives
thereof). Amine-containing polymers such as poly(lysine) (Zauner et
al., 1998, Adv. Drug Del. Rev., 30:97; and Kabanov et al., 1995,
Bioconjugate Chem., 6:7), poly(ethylene imine) (PEI; Boussif et
al., 1995, Proc. Natl. Acad. Sci., USA, 1995, 92:7297), and
poly(amidoamine) dendrimers (Kukowska-Latallo et al., 1996, Proc.
Natl. Acad. Sci., USA, 93:4897; Tang et al., 1996, Bioconjugate
Chem., 7:703; and Haensler et al., 1993, Bioconjugate Chem., 4:372)
are positively-charged at physiological pH, form ion pairs with
nucleic acids, and mediate transfection in a variety of cell lines.
In embodiments, the synthetic nanocarriers may not comprise (or may
exclude) cationic polymers.
In some embodiments, polymers can be degradable polyesters bearing
cationic side chains (Putnam et al., 1999, Macromolecules, 32:3658;
Barrera et al., 1993, J. Am. Chem. Soc., 115:11010; Kwon et al.,
1989, Macromolecules, 22:3250; Lim et al., 1999, J. Am. Chem. Soc.,
121:5633; and Zhou et al., 1990, Macromolecules, 23:3399). Examples
of these polyesters include poly(L-lactide-co-L-lysine) (Barrera et
al., 1993, J. Am. Chem. Soc., 115:11010), poly(serine ester) (Zhou
et al., 1990, Macromolecules, 23:3399), poly(4-hydroxy-L-proline
ester) (Putnam et al., 1999, Macromolecules, 32:3658; and Lim et
al., 1999, J. Am. Chem. Soc., 121:5633), and
poly(4-hydroxy-L-proline ester) (Putnam et al., 1999,
Macromolecules, 32:3658; and Lim et al., 1999, J. Am. Chem. Soc.,
121:5633).
The properties of these and other polymers and methods for
preparing them are well known in the art (see, for example, U.S.
Pat. Nos. 6,123,727; 5,804,178; 5,770,417; 5,736,372; 5,716,404;
6,095,148; 5,837,752; 5,902,599; 5,696,175; 5,514,378; 5,512,600;
5,399,665; 5,019,379; 5,010,167; 4,806,621; 4,638,045; and
4,946,929; Wang et al., 2001, J. Am. Chem. Soc., 123:9480; Lim et
al., 2001, J. Am. Chem. Soc., 123:2460; Langer, 2000, Ace. Chem.
Res., 33:94; Langer, 1999, J. Control. Release, 62:7; and Uhrich et
al., 1999, Chem. Rev., 99:3181). More generally, a variety of
methods for synthesizing certain suitable polymers are described in
Concise Encyclopedia of Polymer Science and Polymeric Amines and
Ammonium Salts, Ed. by Goethals, Pergamon Press, 1980; Principles
of Polymerization by Odian, John Wiley & Sons, Fourth Edition,
2004; Contemporary Polymer Chemistry by Allcock et al.,
Prentice-Hall, 1981; Deming et al., 1997, Nature, 390:386; and in
U.S. Pat. Nos. 6,506,577, 6,632,922, 6,686,446, and 6,818,732.
In some embodiments, polymers can be linear or branched polymers.
In some embodiments, polymers can be dendrimers. In some
embodiments, polymers can be substantially cross-linked to one
another. In some embodiments, polymers can be substantially free of
cross-links. In some embodiments, polymers can be used in
accordance with the present invention without undergoing a
cross-linking step. It is further to be understood that synthetic
nanocarriers may comprise block copolymers, graft copolymers,
blends, mixtures, and/or adducts of any of the foregoing and other
polymers. Those skilled in the art will recognize that the polymers
listed herein represent an exemplary, not comprehensive, list of
polymers that can be of use in accordance with the present
invention.
In some embodiments, synthetic nanocarriers may optionally comprise
one or more amphiphilic entities. In some embodiments, an
amphiphilic entity can promote the production of synthetic
nanocarriers with increased stability, improved uniformity, or
increased viscosity. In some embodiments, amphiphilic entities can
be associated with the interior surface of a lipid membrane (e.g.,
lipid bilayer, lipid monolayer, etc.). Many amphiphilic entities
known in the art are suitable for use in making synthetic
nanocarriers in accordance with the present invention. Such
amphiphilic entities include, but are not limited to,
phosphoglycerides; phosphatidylcholines; dipalmitoyl
phosphatidylcholine (DPPC); dioleylphosphatidyl ethanolamine
(DOPE); dioleyloxypropyltriethylammonium (DOTMA);
dioleoylphosphatidylcholine; cholesterol; cholesterol ester;
diacylglycerol; diacylglycerolsuccinate; diphosphatidyl glycerol
(DPPG); hexanedecanol; fatty alcohols such as polyethylene glycol
(PEG); polyoxyethylene-9-lauryl ether; a surface active fatty acid,
such as palmitic acid or oleic acid; fatty acids; fatty acid
monoglycerides; fatty acid diglycerides; fatty acid amides;
sorbitan trioleate (Span.RTM.85) glycocholate; sorbitan monolaurate
(Span.RTM.20); polysorbate 20 (Tween.RTM.20); polysorbate 60
(Tween.RTM.60); polysorbate 65 (Tween.RTM.65); polysorbate 80
(Tween.RTM.80); polysorbate 85 (Tween.RTM.85); polyoxyethylene
monostearate; surfactin; a poloxomer; a sorbitan fatty acid ester
such as sorbitan trioleate; lecithin; lysolecithin;
phosphatidylserine; phosphatidylinositol; sphingomyelin;
phosphatidylethanolamine (cephalin); cardiolipin; phosphatidic
acid; cerebrosides; dicetylphosphate;
dipalmitoylphosphatidylglycerol; stearylamine; dodecylamine;
hexadecyl-amine; acetyl palmitate; glycerol ricinoleate; hexadecyl
sterate; isopropyl myristate; tyloxapol; poly(ethylene
glycol)5000-phosphatidylethanolamine; poly(ethylene
glycol)-400-monostearate; phospholipids; synthetic and/or natural
detergents having high surfactant properties; deoxycholates;
cyclodextrins; chaotropic salts; ion pairing agents; and
combinations thereof. An amphiphilic entity component may be a
mixture of different amphiphilic entities. Those skilled in the art
will recognize that this is an exemplary, not comprehensive, list
of substances with surfactant activity. Any amphiphilic entity may
be used in the production of synthetic nanocarriers to be used in
accordance with the present invention.
In some embodiments, synthetic nanocarriers may optionally comprise
one or more carbohydrates. Carbohydrates may be natural or
synthetic. A carbohydrate may be a derivatized natural
carbohydrate. In certain embodiments, a carbohydrate comprises
monosaccharide or disaccharide, including but not limited to
glucose, fructose, galactose, ribose, lactose, sucrose, maltose,
trehalose, cellbiose, mannose, xylose, arabinose, glucoronic acid,
galactoronic acid, mannuronic acid, glucosamine, galatosamine, and
neuramic acid. In certain embodiments, a carbohydrate is a
polysaccharide, including but not limited to pullulan, cellulose,
microcrystalline cellulose, hydroxypropyl methylcellulose (HPMC),
hydroxycellulose (HC), methylcellulose (MC), dextran, cyclodextran,
glycogen, hydroxyethylstarch, carageenan, glycon, amylose,
chitosan, N,O-carboxylmethylchitosan, algin and alginic acid,
starch, chitin, inulin, konjac, glucommannan, pustulan, heparin,
hyaluronic acid, curdlan, and xanthan. In embodiments, the
synthetic nanocarriers do not comprise (or specifically exclude)
carbohydrates, such as a polysaccharide. In certain embodiments,
the carbohydrate may comprise a carbohydrate derivative such as a
sugar alcohol, including but not limited to mannitol, sorbitol,
xylitol, erythritol, maltitol, and lactitol.
Compositions for use in the methods according to the invention
comprise synthetic nanocarriers in combination with
pharmaceutically acceptable excipients, such as preservatives,
buffers, saline, or phosphate buffered saline. The compositions may
be made using conventional pharmaceutical manufacturing and
compounding techniques to arrive at useful dosage forms. In an
embodiment, synthetic nanocarriers are suspended in sterile saline
solution for injection together with a preservative.
In embodiments, when preparing synthetic nanocarriers as carriers
for use in vaccines, methods for coupling the components to the
synthetic nanocarriers may be useful. If the component is a small
molecule it may be of advantage to attach the component to a
polymer prior to the assembly of the synthetic nanocarriers. In
embodiments, it may also be an advantage to prepare the synthetic
nanocarriers with surface groups that are used to couple the
component to the synthetic nanocarrier through the use of these
surface groups rather than attaching the component to a polymer and
then using this polymer conjugate in the construction of synthetic
nanocarriers.
In certain embodiments, the coupling can be a covalent linker. In
embodiments, components according to the invention can be
covalently coupled to the external surface via a 1,2,3-triazole
linker formed by the 1,3-dipolar cycloaddition reaction of azido
groups on the surface of the nanocarrier with the component
containing an alkyne group or by the 1,3-dipolar cycloaddition
reaction of alkynes on the surface of the nanocarrier with
components containing an azido group. Such cycloaddition reactions
are preferably performed in the presence of a Cu(I) catalyst along
with a suitable Cu(I)-ligand and a reducing agent to reduce Cu(II)
compound to catalytic active Cu(I) compound. This Cu(I)-catalyzed
azide-alkyne cycloaddition (CuAAC) can also be referred as the
click reaction.
Additionally, the covalent coupling may comprise a covalent linker
that comprises an amide linker, a disulfide linker, a thioether
linker, a hydrazone linker, a hydrazide linker, an imine or oxime
linker, an urea or thiourea linker, an amidine linker, an amine
linker, and a sulfonamide linker.
An amide linker is formed via an amide bond between an amine on one
component with the carboxylic acid group of a second component such
as the nanocarrier. The amide bond in the linker can be made using
any of the conventional amide bond forming reactions with suitably
protected amino acids or antigens or adjuvants and activated
carboxylic acid such N-hydroxysuccinimide-activated ester.
A disulfide linker is made via the formation of a disulfide (S--S)
bond between two sulfur atoms of the form, for instance, of
R1-S--S-R2. A disulfide bond can be formed by thiol exchange of an
antigen or adjuvant containing thiol/mercaptan group (--SH) with
another activated thiol group on a polymer or nanocarrier or a
nanocarrier containing thiol/mercaptan groups with a component
containing activated thiol group.
A triazole linker, specifically a 1,2,3-triazole of the form
##STR00001## wherein R1 and R2 may be any chemical entities, is
made by the 1,3-dipolar cycloaddition reaction of an azide attached
to a first component such as the nanocarrier with a terminal alkyne
attached to a second component. The 1,3-dipolar cycloaddition
reaction is performed with or without a catalyst, preferably with
Cu(I)-catalyst, which links the two components through a
1,2,3-triazole function. This chemistry is described in detail by
Sharpless et al., Angew. Chem. Int. Ed. 41(14), 2596, (2002) and
Meldal, et al, Chem. Rev., 2008, 108(8), 2952-3015 and is often
referred to as a "click" reaction or CuAAC.
In embodiments, a polymer containing an azide or alkyne group,
terminal to the polymer chain is prepared. This polymer is then
used to prepare a synthetic nanocarrier in such a manner that a
plurality of the alkyne or azide groups are positioned on the
surface of that nanocarrier. Alternatively, the synthetic
nanocarrier can be prepared by another route, and subsequently
functionalized with alkyne or azide groups. The component is
prepared with the presence of either an alkyne (if the polymer
contains an azide) or an azide (if the polymer contains an alkyne)
group. The component is then allowed to react with the nanocarrier
via the 1,3-dipolar cycloaddition reaction with or without a
catalyst which covalently couples the component to the particle
through the 1,4-disubstituted 1,2,3-triazole linker.
A thioether linker is made by the formation of a sulfur-carbon
(thioether) bond in the form, for instance, of R1-S-R2. Thioether
can be made by either alkylation of a thiol/mercaptan (--SH) group
on one component with an alkylating group such as halide or epoxide
on a second component such as the nanocarrier. Thioether linkers
can also be formed by Michael addition of a thiol/mercaptan group
on one component to an electron-deficient alkene group on a second
component such as a polymer containing a maleimide group or vinyl
sulfone group as the Michael acceptor. In another way, thioether
linkers can be prepared by the radical thiol-ene reaction of a
thiol/mercaptan group on one component with an alkene group on a
second component such as a polymer or nanocarrier.
A hydrazone linker is made by the reaction of a hydrazide group on
one component with an aldehyde/ketone group on the second component
such as the nanocarrier.
A hydrazide linker is formed by the reaction of a hydrazine group
on one component with a carboxylic acid group on the second
component such as the nanocarrier. Such reaction is generally
performed using chemistry similar to the formation of amide bond
where the carboxylic acid is activated with an activating
reagent.
An imine or oxime linker is formed by the reaction of an amine or
N-alkoxyamine (or aminooxy) group on one component with an aldehyde
or ketone group on the second component such as the
nanocarrier.
An urea or thiourea linker is prepared by the reaction of an amine
group on one component with an isocyanate or thioisocyanate group
on the second component such as the nanocarrier.
An amidine linker is prepared by the reaction of an amine group on
one component with an imidoester group on the second component such
as the nanocarrier.
An amine linker is made by the alkylation reaction of an amine
group on one component with an alkylating group such as halide,
epoxide, or sulfonate ester group on the second component such as
the nanocarrier. Alternatively, an amine linker can also be made by
reductive amination of an amine group on one component with an
aldehyde or ketone group on the second component such as the
nanocarrier with a suitable reducing reagent such as sodium
cyanoborohydride or sodium triacetoxyborohydride.
A sulfonamide linker is made by the reaction of an amine group on
one component with a sulfonyl halide (such as sulfonyl chloride)
group on the second component such as the nanocarrier.
A sulfone linker is made by Michael addition of a nucleophile to a
vinyl sulfone. Either the vinyl sulfone or the nucleophile may be
on the surface of the nanocarrier or attached to a component.
The component can also be conjugated to the nanocarrier via
non-covalent conjugation methods. For examples, a negative charged
component can be conjugated to a positive charged nanocarrier
through electrostatic adsorption. A component containing a metal
ligand can also be conjugated to a nanocarrier containing a metal
complex via a metal-ligand complex.
In embodiments, the component can be attached to a polymer, for
example polylactic acid-block-polyethylene glycol, prior to the
assembly of the synthetic nanocarrier or the synthetic nanocarrier
can be formed with reactive or activatable groups on its surface.
In the latter case, the component may be prepared with a group
which is compatible with the attachment chemistry that is presented
by the synthetic nanocarriers' surface. In other embodiments, a
component can be attached to VLPs or liposomes using a suitable
linker. A linker is a compound or reagent that capable of coupling
two molecules together. In an embodiment, the linker can be a
homobifuntional or heterobifunctional reagent as described in
Hermanson 2008. For example, an VLP or liposome synthetic
nanocarrier containing a carboxylic group on the surface can be
treated with a homobifunctional linker, adipic dihydrazide (ADH),
in the presence of EDC to form the corresponding synthetic
nanocarrier with the ADH linker. The resulting ADH linked synthetic
nanocarrier is then conjugated with a component containing an acid
group via the other end of the ADH linker on NC to produce the
corresponding VLP or liposome peptide conjugate.
For detailed descriptions of available conjugation methods, see
Hermanson G T "Bioconjugate Techniques", 2nd Edition Published by
Academic Press, Inc., 2008. In addition to covalent attachment the
component can be coupled by adsorption to a pre-formed synthetic
nanocarrier or it can be coupled by encapsulation during the
formation of the synthetic nanocarrier.
In some embodiments, a component, such as an antigen or adjuvant,
may be isolated. Isolated refers to the element being separated
from its native environment and present in sufficient quantities to
permit its identification or use. This means, for example, the
element may be (i) selectively produced by expression cloning or
(ii) purified as by chromatography or electrophoresis. Isolated
elements may be, but need not be, substantially pure. Because an
isolated element may be admixed with a pharmaceutically acceptable
excipient in a pharmaceutical preparation, the element may comprise
only a small percentage by weight of the preparation. The element
is nonetheless isolated in that it has been separated from the
substances with which it may be associated in living systems, i.e.,
isolated from other lipids or proteins. Any of the elements
provided herein may be isolated. Any of the antigens provided
herein can be included in the compositions in isolated form.
D. Methods of Using and Making Synthetic Nanocarrier
Compositions
Synthetic nanocarriers may be prepared using a wide variety of
methods known in the art. For example, synthetic nanocarriers can
be formed by methods as nanoprecipitation, flow focusing fluidic
channels, spray drying, single and double emulsion solvent
evaporation, solvent extraction, phase separation, milling,
microemulsion procedures, microfabrication, nanofabrication,
sacrificial layers, simple and complex coacervation, and other
methods well known to those of ordinary skill in the art.
Alternatively or additionally, aqueous and organic solvent
syntheses for monodisperse semiconductor, conductive, magnetic,
organic, and other nanomaterials have been described (Pellegrino et
al., 2005, Small, 1:48; Murray et al., 2000, Ann. Rev. Mat. Sci.,
30:545; and Trindade et al., 2001, Chem. Mat., 13:3843). Additional
methods have been described in the literature (see, e.g., Doubrow,
Ed., "Microcapsules and Nanoparticles in Medicine and Pharmacy,"
CRC Press, Boca Raton, 1992; Mathiowitz et al., 1987, J. Control.
Release, 5:13; Mathiowitz et al., 1987, Reactive Polymers, 6: 275;
and Mathiowitz et al., 1988, J. Appl. Polymer Sci., 35:755; U.S.
Pat. Nos. 5,578,325 and 6,007,845; P. Paolicelli et al.,
"Surface-modified PLGA-based Nanoparticles that can Efficiently
Associate and Deliver Virus-like Particles" Nanomedicine.
5(6):843-853 (2010)).
Various materials may be encapsulated into synthetic nanocarriers
as desirable using a variety of methods including but not limited
to C. Astete et al., "Synthesis and characterization of PLGA
nanoparticles" J. Biomater. Sci. Polymer Edn, Vol. 17, No. 3, pp.
247-289 (2006); K. Avgoustakis "Pegylated Poly(Lactide) and
Poly(Lactide-Co-Glycolide) Nanoparticles: Preparation, Properties
and Possible Applications in Drug Delivery" Current Drug Delivery
1:321-333 (2004); C. Reis et al., "Nanoencapsulation I. Methods for
preparation of drug-loaded polymeric nanoparticles" Nanomedicine
2:8-21 (2006); P. Paolicelli et al., "Surface-modified PLGA-based
Nanoparticles that can Efficiently Associate and Deliver Virus-like
Particles" Nanomedicine. 5(6):843-853 (2010). Other methods
suitable for encapsulating materials into synthetic nanocarriers
may be used, including without limitation methods disclosed in U.S.
Pat. No. 6,632,671 to Unger Oct. 14, 2003.
In certain embodiments, synthetic nanocarriers are prepared by a
nanoprecipitation process or spray drying. Conditions used in
preparing synthetic nanocarriers may be altered to yield particles
of a desired size or property (e.g., hydrophobicity,
hydrophilicity, external morphology, "stickiness," shape, etc.).
The method of preparing the synthetic nanocarriers and the
conditions (e.g., solvent, temperature, concentration, air flow
rate, etc.) used may depend on the materials to be coupled to the
synthetic nanocarriers and/or the composition of the polymer
matrix.
If particles prepared by any of the above methods have a size range
outside of the desired range, particles can be sized, for example,
using a sieve.
Elements of the synthetic nanocarriers may be coupled to the
overall synthetic nanocarrier, e.g., by one or more covalent bonds,
or may be coupled by means of one or more linkers. Additional
methods of functionalizing synthetic nanocarriers may be adapted
from Published US Patent Application 2006/0002852 to Saltzman et
al., Published US Patent Application 2009/0028910 to DeSimone et
al., or Published International Patent Application WO/2008/127532
A1 to Murthy et al.
Alternatively or additionally, synthetic nanocarriers can be
coupled to elements directly or indirectly via non-covalent
interactions. In non-covalent embodiments, the non-covalent
coupling is mediated by non-covalent interactions including but not
limited to charge interactions, affinity interactions, metal
coordination, physical adsorption, host-guest interactions,
hydrophobic interactions, TT stacking interactions, hydrogen
bonding interactions, van der Waals interactions, magnetic
interactions, electrostatic interactions, dipole-dipole
interactions, and/or combinations thereof. Such couplings may be
arranged to be on an external surface or an internal surface of an
synthetic nanocarrier. In embodiments, encapsulation and/or
absorption is a form of coupling.
In embodiments, the synthetic nanocarriers can be combined with
adjuvants by admixing in the same vehicle or delivery system. Such
adjuvants may include, but are not limited to mineral salts, such
as alum, alum combined with monphosphoryl lipid (MPL) A of
Enterobacteria, such as Escherihia coli, Salmonella minnesota,
Salmonella typhimurium, or Shigella flexneri or specifically with
MPL.RTM. (AS04), MPL A of above-mentioned bacteria separately,
saponins, such as QS-21, Quil-A, ISCOMs, ISCOMATRIX.TM., emulsions
such as MF59.TM., Montanide.RTM. ISA 51 and ISA 720, AS02
(QS21+squalene+MPL.RTM.), liposomes and liposomal formulations such
as AS01, synthesized or specifically prepared microparticles and
microcarriers such as bacteria-derived outer membrane vesicles
(OMV) of N. gonorrheae, Chlamydia trachomatis and others, or
chitosan particles, depot-forming agents, such as Pluronic.RTM.
block co-polymers, specifically modified or prepared peptides, such
as muramyl dipeptide, aminoalkyl glucosaminide 4-phosphates, such
as RC529, or proteins, such as bacterial toxoids or toxin
fragments. The doses of such other adjuvants can be determined
using conventional dose ranging studies.
In embodiments, the synthetic nanocarriers can be combined with an
antigen different, similar or identical to those coupled to a
nanocarrier (with or without adjuvant, utilizing or not utilizing
another delivery vehicle) administered separately at a different
time-point and/or at a different body location and/or by a
different immunization route or with another antigen and/or
adjuvant-carrying synthetic nanocarrier administered separately at
a different time-point and/or at a different body location and/or
by a different immunization route.
Populations of synthetic nanocarriers may be combined to form
pharmaceutical dosage forms according to the present invention
using traditional pharmaceutical mixing methods. These include
liquid-liquid mixing in which two or more suspensions, each
containing one or more subsets of nanocarriers, are directly
combined or are brought together via one or more vessels containing
diluent. As synthetic nanocarriers may also be produced or stored
in a powder form, dry powder-powder mixing could be performed as
could the re-suspension of two or more powders in a common media.
Depending on the properties of the nanocarriers and their
interaction potentials, there may be advantages conferred to one or
another route of mixing.
Typical compositions that comprise synthetic nanocarriers may
comprise inorganic or organic buffers (e.g., sodium or potassium
salts of phosphate, carbonate, acetate, or citrate) and pH
adjustment agents (e.g., hydrochloric acid, sodium or potassium
hydroxide, salts of citrate or acetate, amino acids and their
salts) antioxidants (e.g., ascorbic acid, alpha-tocopherol),
surfactants (e.g., polysorbate 20, polysorbate 80,
polyoxyethylene9-10 nonyl phenol, sodium desoxycholate), solution
and/or cryo/lyo stabilizers (e.g., sucrose, lactose, mannitol,
trehalose), osmotic adjustment agents (e.g., salts or sugars),
antibacterial agents (e.g., benzoic acid, phenol, gentamicin),
antifoaming agents (e.g., polydimethylsilozone), preservatives
(e.g., thimerosal, 2-phenoxyethanol, EDTA), polymeric stabilizers
and viscosity-adjustment agents (e.g., polyvinylpyrrolidone,
poloxamer 488, carboxymethylcellulose) and co-solvents (e.g.,
glycerol, polyethylene glycol, ethanol).
Compositions according to the invention comprise synthetic
nanocarriers in combination with pharmaceutically acceptable
excipients. The compositions may be made using conventional
pharmaceutical manufacturing and compounding techniques to arrive
at useful dosage forms. Techniques suitable for use in practicing
the present invention may be found in Handbook of Industrial
Mixing: Science and Practice, Edited by Edward L. Paul, Victor A.
Atiemo-Obeng, and Suzanne M. Kresta, 2004 John Wiley & Sons,
Inc.; and Pharmaceutics: The Science of Dosage Form Design, 2nd Ed.
Edited by M. E. Auten, 2001, Churchill Livingstone. In an
embodiment, synthetic nanocarriers are suspended in sterile saline
solution for injection together with a preservative.
It is to be understood that the compositions of synthetic
nanocarriers can be made in any suitable manner, and the invention
is in no way limited to the use of compositions that can be
produced using the methods described herein. Selection of an
appropriate method may require attention to the properties of the
particular elements being associated.
In some embodiments, synthetic nanocarriers are manufactured under
sterile conditions or are terminally sterilized. This can ensure
that resulting composition are sterile and non-infectious, thus
improving safety when compared to non-sterile compositions. This
provides a valuable safety measure, especially when subjects
receiving synthetic nanocarriers have immune defects, are suffering
from infection, and/or are susceptible to infection. In some
embodiments, synthetic nanocarriers may be lyophilized and stored
in suspension or as lyophilized powder depending on the formulation
strategy for extended periods without losing activity.
The compositions of the invention can be administered by a variety
of routes, including or not limited to subcutaneous, intranasal,
oral, intravenous, intraperitoneal, intramuscular, transmucosal,
transmucosal, sublingual, rectal, ophthalmic, pulmonary,
intradermal, transdermal, transcutaneous or intradermal or by a
combination of these routes. Routes of administration also include
administration by inhalation or pulmonary aerosol. Techniques for
preparing aerosol delivery systems are well known to those of skill
in the art (see, for example, Sciarra and Cutie, "Aerosols," in
Remington's Pharmaceutical Sciences, 18th edition, 1990, pp.
1694-1712; incorporated by reference).
Doses of dosage forms contain varying amounts of populations of
synthetic nanocarriers and varying amounts of the proteins and/or
adjuvants and/or additional antigens, according to the invention.
The amount of synthetic nanocarriers and/or proteins and/or
adjuvants and/or additional antigens present in the dosage forms
can be varied according to the nature of the elements present, the
therapeutic benefit to be accomplished, and other such parameters.
In embodiments, dose ranging studies can be conducted to establish
optimal therapeutic amount of the population of synthetic
nanocarriers and the amount of proteins and/or adjuvants and/or
additional antigens to be present in the dosage form. In
embodiments, the synthetic nanocarriers and the proteins and/or
adjuvants and/or additional antigens are present in the dosage form
in an amount effective to generate an immune response to the
proteins and/or additional antigens upon administration to a
subject. It may be possible to determine amounts effective to
generate an immune response using conventional dose ranging studies
and techniques in subjects. Dosage forms may be administered at a
variety of frequencies. In a preferred embodiment, at least one
administration of the dosage form is sufficient to generate a
pharmacologically relevant response. In more preferred embodiment,
at least two administrations, at least three administrations, or at
least four administrations, of the dosage form are utilized to
ensure a pharmacologically relevant response.
The compositions and methods described herein can be used to
induce, enhance, suppress, modulate, direct, or redirect an immune
response. The compositions and methods described herein can be used
in the diagnosis, prophylaxis and/or treatment of conditions such
as cancers, infectious diseases, metabolic diseases, degenerative
diseases, non-autoimmune diseases, HIV, malaria, hepatitis B or any
of the other disorders and/or conditions provided herein.
Examples of infectious disease include, but are not limited to,
viral infectious diseases, such as AIDS, Chickenpox (Varicella),
Common cold, Cytomegalovirus Infection, Colorado tick fever, Dengue
fever, Ebola hemorrhagic fever, Hand, foot and mouth disease,
Hepatitis, Herpes simplex, Herpes zoster, HPV, Influenza (Flu),
Lassa fever, Measles, Marburg hemorrhagic fever, Infectious
mononucleosis, Mumps, Norovirus, Poliomyelitis, Progressive
multifocal leukencephalopathy, Rabies, Rubella, SARS, Smallpox
(Variola), Viral encephalitis, Viral gastroenteritis, Viral
meningitis, Viral pneumonia, West Nile disease and Yellow fever;
bacterial infectious diseases, such as Anthrax, Bacterial
Meningitis, Botulism, Brucellosis, Campylobacteriosis, Cat Scratch
Disease, Cholera, Diphtheria, Epidemic Typhus, Gonorrhea, Impetigo,
Legionellosis, Leprosy (Hansen's Disease), Leptospirosis,
Listeriosis, Lyme disease, Melioidosis, Rheumatic Fever, MRSA
infection, Nocardiosis, Pertussis (Whooping Cough), Plague,
Pneumococcal pneumonia, Psittacosis, Q fever, Rocky Mountain
Spotted Fever (RMSF), Salmonellosis, Scarlet Fever, Shigellosis,
Syphilis, Tetanus, Trachoma, Tuberculosis, Tularemia, Typhoid
Fever, Typhus and Urinary Tract Infections; parasitic infectious
diseases, such as African trypanosomiasis, Amebiasis, Ascariasis,
Babesiosis, Chagas Disease, Clonorchiasis, Cryptosporidiosis,
Cysticercosis, Diphyllobothriasis, Dracunculiasis, Echinococcosis,
Enterobiasis, Fascioliasis, Fasciolopsiasis, Filariasis,
Free-living amebic infection, Giardiasis, Gnathostomiasis,
Hymenolepiasis, Isosporiasis, Kala-azar, Leishmaniasis, Malaria,
Metagonimiasis, Myiasis, Onchocerciasis, Pediculosis, Pinworm
Infection, Scabies, Schistosomiasis, Taeniasis, Toxocariasis,
Toxoplasmosis, Trichinellosis, Trichinosis, Trichuriasis,
Trichomoniasis and Trypanosomiasis; fungal infectious disease, such
as Aspergillosis, Blastomycosis, Candidiasis, Coccidioidomycosis,
Cryptococcosis, Histoplasmosis, Tinea pedis (Athlete's Foot) and
Tinea cruris; prion infectious diseases, such as Alpers' disease,
Fatal Familial Insomnia, Gerstmann-Straiussler-Scheinker syndrome,
Kuru and Variant Creutzfeldt-Jakob disease.
Examples of cancers include, but are not limited to breast cancer;
biliary tract cancer; bladder cancer; brain cancer including
glioblastomas and medulloblastomas; cervical cancer;
choriocarcinoma; colon cancer; endometrial cancer; esophageal
cancer; gastric cancer; hematological neoplasms including acute
lymphocytic and myelogenous leukemia, e.g., B Cell CLL; T-cell
acute lymphoblastic leukemia/lymphoma; hairy cell leukemia; chronic
myelogenous leukemia, multiple myeloma; AIDS-associated leukemias
and adult T-cell leukemia/lymphoma; intraepithelial neoplasms
including Bowen's disease and Paget's disease; liver cancer; lung
cancer; lymphomas including Hodgkin's disease and lymphocytic
lymphomas; neuroblastomas; oral cancer including squamous cell
carcinoma; ovarian cancer including those arising from epithelial
cells, stromal cells, germ cells and mesenchymal cells; pancreatic
cancer; prostate cancer; rectal cancer; sarcomas including
leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and
osteosarcoma; skin cancer including melanoma, Merkel cell
carcinoma, Kaposi's sarcoma, basal cell carcinoma, and squamous
cell cancer; testicular cancer including germinal tumors such as
seminoma, non-seminoma (teratomas, choriocarcinomas), stromal
tumors, and germ cell tumors; thyroid cancer including thyroid
adenocarcinoma and medullar carcinoma; and renal cancer including
adenocarcinoma and Wilms tumor.
Examples of metabolic diseases include, but are not limited to,
disorders of carbohydrate metabolism, amino acid metabolism,
organic acid metabolism, fatty acid oxidation and mitochondrial
metabolism, prophyrin metabolism, purine or pyrimidine metabolism,
steroid metabolism, lysosomal mitochondrial function, peroxisomal
function, lysosomal storage, urea cycle disorders (e.g., N-acetyl
glutamate synthetase deficiency, carbamylphosphate synthase
deficiency, ornithine carbamyl transferase deficiency,
crginosuccinic aciduria, citrullinaemia, arginase deficiency),
amino acid disorders (e.g., Non-ketotic hyperglycinaemia,
tyrosinaemia (Type I), Maple syrup urine disease), organic
acidemias (e.g, isovaleric acidemia, methylmalonic acidemia,
propionic acidemia, glutaric aciduria type I, glutaric acidemia
type I & II), mitochondrial disorders (e.g., carboxylase
defects, mitochondrial myopathies, lactic acidosis (pyruvate
dehydrogenase complex defects), congenital lactic acidosis,
mitochondrial respiratory chain defects, cystinosis, Gaucher's
disease, Fabry's disease, Pompe's disease, mucopolysaccharoidosis
I, mucopolysaccharoidosis II, mucopolysaccharoidosis VI).
Examples of degenerative diseases include, but are not limited to,
mesenchyme/mesoderm degenerative disease, muscle degenerative
disease, endothelial degenerative disease, neurodegenerative
disease, degenerative joint disease (e.g., osteoarthritis), major
types of degenerative heart disease (e.g., coronary heart disease,
congenital heart disease, rheumatic heart disease, angina
pectoris), neurodegenerative disease (e.g., Alzheimer's disease,
amyotrophic lateral sclerosis, Friedreich's ataxia, Huntington's
disease, Lewy body disease, Parkinson's disease, spinal muscular
atrophy), neuromuscular disorders (e.g., muscular dystrophy,
duchenne muscular dystrophy, facioscapulohumeral muscular
dystrophy, myotonic muscular dystrophy, congenital myopathy,
familial cardiomyopathy, dilated cardiomyopathy, hypertrophic
cardiomyopathy, restrictive cardiomyopathy, or coronary artery
disease).
The proteins for coupling to the synthetic nanocarriers and/or the
additional antigens provided herein can be antigens associated with
any of the diseases or conditions provided herein. These include
antigens associated with cancer, infections or infectious disease
or degenerative or non-autoimmune disease. Antigens associated with
HIV, malaria, leischmaniasis, a human filovirus infection, a
togavirus infection, a alphavirus infection, an arenavirus
infection, a bunyavirus infection, a flavivirus infection, a human
papillomavirus infection, a human influenza A virus infection, a
hepatitis B infection or a hepatitis C infection are also
included.
Examples of cancer antigens include HER 2 (p185), CD20, CD33, GD3
ganglioside, GD2 ganglioside, carcinoembryonic antigen (CEA), CD22,
milk mucin core protein, TAG-72, Lewis A antigen, ovarian
associated antigens such as OV-TL3 and MOv18, high Mr melanoma
antigens recognized by antibody 9.2.27, HMFG-2, SM-3, B72.3, PR5C5,
PR4D2, and the like. Further examples include MAGE, MART-1/Melan-A,
gp100, Dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding
protein (ADAbp), FAP, cyclophilin b, Colorectal associated antigen
(CRC)-C017-1A/GA733, Carcinoembryonic Antigen (CEA) and its
immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, prostatic acid
phosphatase (PAP), Prostate Specific Antigen (PSA) and its
immunogenic epitopes PSA-1, PSA-2, and PSA-3, prostate-specific
membrane antigen (PSMA), T-cell receptor/CD3-zeta chain,
MAGE-family of tumor antigens (e.g., MAGE-I or MAGE-II families)
(e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6,
MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2
(MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1,
MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE-family of tumor antigens
(e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7,
GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4,
tyrosinase, p53, MUC family, HER2/neu, p21ras, RCAS1,
.alpha.-fetoprotein, E-cadherin, .alpha.-catenin, .beta.-catenin
and .gamma.-catenin, p120ctn, gp100Pme1117, PRAME, NY-ESO-1, cdc27,
adenomatous polyposis coli protein (APC), fodrin, Connexin 37,
Ig-idiotype, p15, gp75, GM2 and GD2 gangliosides, viral products
such as human papilloma virus proteins, Smad family of tumor
antigens, Imp-1, P1A, EBV-encoded nuclear antigen (EBNA)-1, brain
glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4,
SSX-5, SCP-1 and CT-7, CD20 and c-erbB-2.
In another embodiment, antigens associated with infection or
infectious disease are associated with any of the infectious agents
provided herein. In one embodiment, the infectious agent is a virus
of the Adenoviridae, Picornaviridae, Herpesviridae, Hepadnaviridae,
Flaviviridae, Retroviridae, Orthomyxoviridae, Paramyxoviridae,
Papillomaviridae, Rhabdoviridae, Togaviridae or Paroviridae family.
In still another embodiment, the infectious agent is adenovirus,
coxsackievirus, hepatitis A virus, poliovirus, Rhinovirus, Herpes
simplex virus, Varicella-zoster virus, Epstein-barr virus, Human
cytomegalovirus, Human herpesvirus, Hepatitis B virus, Hepatitis C
virus, yellow fever virus, dengue virus, West Nile virus, HIV,
Influenza virus, Measles virus, Mumps virus, Parainfluenza virus,
Respiratory syncytial virus, Human metapneumovirus, Human
papillomavirus, Rabies virus, Rubella virus, Human bocarivus or
Parvovirus B 19. In yet another embodiment, the infectious agent is
a bacteria of the Bordetella, Borrelia, Brucella, Campylobacter,
Chlamydia and Chlamydophila, Clostridium, Corynebacterium,
Enterococcus, Escherichia, Francisella, Haemophilus, Helicobacter,
Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma,
Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella,
Staphylococcus, Streptococcus, Treponema Vibrio or Yersinia genus.
In a further embodiment, the infectious agent is Bordetella
pertussis, Borrelia burgdorferi, Brucella abortus, Brucella canis,
Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia
pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci,
Clostridium botulinum, Clostridium difficile, Clostridium
perfringens, Clostridium tetani, Corynebacterium diphtheriae,
Enterococcus faecalis, Enterococcus faecium, Escherichia coli,
Francisella tularensis, Haemophilus influenzae, Helicobacter
pylori, Legionella pneumophila, Leptospira interrogans, Listeria
monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis,
Mycobacterium ulcerans, Mycoplasma pneumoniae, Neisseria
gonorrhoeae, Neisseria meningitides, Pseudomonas aeruginosa,
Rickettsia rickettsii, Salmonella typhi, Salmonella typhimurium,
Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermidis,
Staphylococcus saprophyticus, Streptococcus agalactiae,
Streptococcus pneumoniae, Streptococcus pyogenes, Treponema
pallidum, Vibrio cholerae or Yersinia pestis. In another
embodiment, the infectious agent is a fungus of the Candida,
Aspergillus, Cryptococcus, Histoplasma, Pneumocystis or
Stachybotrys genus. In still another embodiment, the infectious
agent is C. albicans, Aspergillus fumigatus, Aspergillus flavus,
Cryptococcus neoformans, Cryptococcus laurentii, Cryptococcus
albidus, Cryptococcus gattii, Histoplasma capsulatum, Pneumocystis
jirovecii or Stachybotrys chartarum.
In yet another embodiment, the antigen associated with infection or
infectious disease is one that comprises VI, VII, E1A, E3-19K, 52K,
VP1, surface antigen, 3A protein, capsid protein, nucleocapsid,
surface projection, transmembrane proteins, UL6, UL18, UL35, UL38,
UL19, early antigen, capsid antigen, Pp65, gB, p52, latent nuclear
antigen-1, NS3, envelope protein, envelope protein E2 domain,
gp120, p24, lipopeptides Gag (17-35), Gag (253-284), Nef (66-97),
Nef (116-145), Pol (325-355), neuraminidase, nucleocapsid protein,
matrix protein, phosphoprotein, fusion protein, hemagglutinin,
hemagglutinin-neuraminidase, glycoprotein, E6, E7, envelope
lipoprotein or non-structural protein (NS). In another embodiment,
the antigen comprises pertussis toxin (PT), filamentous
hemagglutinin (FHA), pertactin (PRN), fimbriae (FIM 2/3), VlsE;
DbpA, OspA, Hia, PrpA, MltA, L7/L12, D15, 0187, VirJ, Mdh, AfuA,
L7/L12, out membrane protein, LPS, antigen type A, antigen type B,
antigen type C, antigen type D, antigen type E, FliC, FliD, Cwp84,
alpha-toxin, theta-toxin, fructose 1,6-biphosphate-aldolase (FBA),
glyceraldehydes-3-phosphate dehydrogenase (GPD),
pyruvate:ferredoxin oxidoreductase (PFOR), elongation factor-G
(EF-G), hypothetical protein (HP), T toxin, Toxoid antigen,
capsular polysaccharide, Protein D, Mip, nucleoprotein (NP), RD1,
PE35, PPE68, EsxA, EsxB, RD9, EsxV, Hsp70, lipopolysaccharide,
surface antigen, Sp1, Sp2, Sp3, Glycerophosphodiester
Phosphodiesterase, outer membrane protein, chaperone-usher protein,
capsular protein (F1) or V protein. In yet another embodiment, the
antigen is one that comprises capsular glycoprotein, Yps3P, Hsp60,
Major surface protein, MsgC1, MsgC3, MsgC8, MsgC9 or SchS34.
EXAMPLES
Example 1
Synthetic Nanocarrier Formulation Lot #1
Materials
Ovalbumin protein, was purchased from Worthington Biochemical
Corporation (730 Vassar Avenue, Lakewood, N.J. 08701. Product Code
3048.) PLGA-R848 of approximately 5,200 Da made from PLGA of 3:1
lactide to glycolide ratio and having 12.7% w/w conjugated R848
content was synthesized. PLA-PEG-Nicotine with a
nicotine-terminated PEG block of approximately 5,000 Da and DL-PLA
block of approximately 19,000 Da was synthesized. PLA with an
inherent viscosity of 0.21 dL/g was purchased from SurModics
Pharmaceuticals (756 Tom Martin Drive, Birmingham, Ala. 35211.
Product Code 100 DL 2A.) Polyvinyl alcohol (Mw=11,000-31,000,
87-89% hydrolyzed) was purchased from J. T. Baker (Part Number
U232-08).
Method
Solutions were prepared as follows:
Solution 1: Ovalbumin protein at 20 mg/mL in 10 mM phosphate
buffer.
Solution 2: PLGA-R848 at 50 mg/mL, PLA-PEG-Nicotine at 25 mg/mL,
PLA at 25 mg/ml in dichloromethane. The solution was prepared by
separately dissolving each polymer as a 100 mg/mL in
dichloromethane, then mixing the solutions by adding 2 parts
PLGA-R848 solution to 1 part each PLA-PEG-Nicotine solution and PLA
solution.
Solution 3: Polyvinyl alcohol 50 mg/mL in 100 mM in 100 mM
phosphate buffer, pH 8.
Solution 4: 70 mM phosphate buffer, pH 8.
A primary (W1/O) emulsion was first created using Solution 1 &
Solution 2. Solution 1 (0.2 mL) and Solution 2 (1.0 mL) were
combined in a small glass pressure tube and sonicated at 50%
amplitude for 40 seconds using a Branson Digital Sonifier 250. A
secondary (W1/O/W2) emulsion was then formed by adding Solution 3
(2.0 mL) to the primary emulsion, and then sonicating at 30%
amplitude for 40 seconds using the Branson Digital Sonifier 250.
The secondary emulsion was added to an open 50 mL beaker containing
70 mM phosphate buffer solution (30 mL) and stirred at room
temperature for 2 hours to allow the dichloromethane to evaporate
and the nanocarriers to form in suspension. A portion of the
suspended nanocarriers was washed by transferring the nanocarrier
suspension to a centrifuge tube, spinning at 13,823 g for one hour,
removing the supernatant, and re-suspending the pellet in phosphate
buffered saline. This washing procedure was repeated and then the
pellet was re-suspended in phosphate buffered saline to achieve a
nanocarrier suspension having a nominal concentration of 10 mg/mL
on a polymer basis. The suspension was stored frozen at -20.degree.
C. until use.
TABLE-US-00001 TABLE 1 Nanocarrier Characterization Nanocarrier
Effective R848 Ovalbumin ID Diameter (nm) (% w/w) (% w/w) 1 214 4.0
1.1
Example 2
Synthetic Nanocarrier Formulation Lot #2
Materials
Ovalbumin protein, was purchased from Worthington Biochemical
Corporation (730 Vassar Avenue, Lakewood, N.J. 08701. Product Code
3048.) Ovalbumin peptide 323-339 amide acetate salt, was purchased
from Bachem Americas Inc. (3132 Kashiwa Street, Torrance Calif.
90505. Product code 4065609.) PLGA-R848 of approximately 5,200 Da
made from PLGA of 3:1 lactide to glycolide ratio and having 12.7%
w/w conjugated R848 content was synthesized. PLA-PEG-Nicotine with
a nicotine-terminated PEG block of approximately 5,000 Da and
DL-PLA block of approximately 19,000 Da was synthesized. PLA with
an inherent viscosity of 0.21 dL/g was purchased from SurModics
Pharmaceuticals (756 Tom Martin Drive, Birmingham, Ala. 35211.
Product Code 100 DL 2A.) Polyvinyl alcohol (Mw=11,000-31,000,
87-89% hydrolyzed) was purchased from J. T. Baker (Part Number
U232-08).
Method
Solutions were prepared as follows:
Solution 1A: Ovalbumin protein at 40 mg/mL in 10 mM phosphate
buffer.
Solution 1B: Ovalbumin peptide amide 323-339 @ 40 mg/mL in dilute
hydrochloric acid aqueous solution.
Solution 2: PLGA-R848 at 50 mg/mL, PLA-PEG-Nicotine at 25 mg/mL,
PLA at 25 mg/ml in dichloromethane. The solution was prepared by
separately dissolving each polymer at 100 mg/mL in dichloromethane,
then mixing the solutions by adding 2 parts PLGA-R848 solution to 1
part each PLA-PEG-Nicotine solution and PLA solution.
Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM in 100 mM
phosphate buffer, pH 8.
Solution 4: 70 mM phosphate buffer, pH 8.
The first primary (W1/O) emulsion was created using Solution 1A
& Solution 2. Solution 1A (0.2 mL) and Solution 2 (1.0 mL) were
combined in a small glass pressure tube and sonicated at 50%
amplitude for 40 seconds using a Branson Digital Sonifier 250. A
second primary (W1/O) emulsion was created using Solution 1B &
Solution 2. Solution 1B (0.2 mL) and Solution 2 (1.0 mL) were
combined in a small glass pressure tube and sonicated at 50%
amplitude for 40 seconds using a Branson Digital Sonifier 250.
Approximately 1/2 of the second primary emulsion was removed from
its pressure tube and discarded and then 1/2 of the first primary
emulsion (0.500 mL), was added to the tube to create a 1:1 mixture
of the two primary emulsions in a total volume of approximately 1
mL. A secondary (W1/O/W2) emulsion was then formed by adding
Solution 3 (2.0 mL) to the primary emulsion, and then sonicating at
30% amplitude for 40 seconds using the Branson Digital Sonifier
250. The secondary emulsion was added to an open 50 mL beaker
containing 70 mM phosphate buffer solution (30 mL) and stirred at
room temperature for 2 hours to allow the dichloromethane to
evaporate and the nanocarriers to form in suspension. A portion of
the suspended nanocarriers was washed by transferring the
nanocarrier suspension to a centrifuge tube, spinning at 13,823 g
for one hour, removing the supernatant, and re-suspending the
pellet in phosphate buffered saline. This washing procedure was
repeated and then the pellet was re-suspended in phosphate buffered
saline to achieve a nanocarrier suspension having a nominal
concentration of 10 mg/mL on a polymer basis. The suspension was
stored frozen at -20.degree. C. until use.
TABLE-US-00002 TABLE 2 Nanocarrier Characterization Ovalbumin
Ovalbumin Nanocarrier Effective R848 Protein Peptide ID Diameter
(nm) (% w/w) (% w/w) (% w/w) 2 234 3.9 0.3 2.3
Example 3
Synthetic Nanocarrier Formulation Lot #3
Materials
Ovalbumin protein, was purchased from Worthington Biochemical
Corporation (730 Vassar Avenue, Lakewood, N.J. 08701. Product Code
3048.) PLGA-R848 of approximately 5,200 Da made from PLGA of 3:1
lactide to glycolide ratio and having 12.7% w/w conjugated R848
content was synthesized. PLA-PEG-OMe block co-polymer with a methyl
ether terminated PEG block of 2,000 Da and DL-PLA block of
approximately 19,000 Da was synthesized. PLA with an inherent
viscosity of 0.21 dL/g was purchased from SurModics Pharmaceuticals
(756 Tom Martin Drive, Birmingham, Ala. 35211. Product Code 100 DL
2A.) Polyvinyl alcohol (Mw=11,000-31,000, 87-89% hydrolyzed) was
purchased from J. T. Baker (Part Number U232-08).
Method
Solutions were prepared as follows:
Solution 1: Ovalbumin protein at 20 mg/mL in 10 mM phosphate
buffer.
Solution 2: PLGA-R848 at 50 mg/mL, PLA-PEG-OMe at 25 mg/mL, PLA at
25 mg/ml in dichloromethane. The solution was prepared by
separately dissolving each polymer as a 100 mg/mL in
dichloromethane, then mixing the solutions by adding 2 parts
PLGA-R848 solution to 1 part each PLA-PEG-OMe solution and PLA
solution.
Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM in 100 mM
phosphate buffer, pH 8.
Solution 4: 70 mM phosphate buffer, pH 8.
A primary (W1/O) emulsion was first created using Solution 1 &
Solution 2. Solution 1 (0.2 mL) and Solution 2 (1.0 mL) were
combined in a small glass pressure tube and sonicated at 50%
amplitude for 40 seconds using a Branson Digital Sonifier 250. A
secondary (W1/O/W2) emulsion was then formed by adding Solution 3
(2.0 mL) to the primary emulsion, and then sonicating at 30%
amplitude for 40 seconds using the Branson Digital Sonifier 250.
The secondary emulsion was added to an open 50 mL beaker containing
70 mM phosphate buffer solution (30 mL) and stirred at room
temperature for 2 hours to allow the dichloromethane to evaporate
and the nanocarriers to form in suspension. A portion of the
suspended nanocarriers was washed by transferring the nanocarrier
suspension to a centrifuge tube, spinning at 13,823 g for one hour,
removing the supernatant, and re-suspending the pellet in phosphate
buffered saline. This washing procedure was repeated and then the
pellet was re-suspended in phosphate buffered saline to achieve a
nanocarrier suspension having a nominal concentration of 10 mg/mL
on a polymer basis. The suspension was stored frozen at -20.degree.
C. until use.
TABLE-US-00003 TABLE 3 Nanocarrier Characterization Nanocarrier
Effective R848 Ovalbumin ID Diameter (nm) (% w/w) (% w/w) 3 217 4.3
0.8
Example 4
Synthetic Nanocarrier Formulation Lot #4
Ovalbumin protein, was purchased from Worthington Biochemical
Corporation (730 Vassar Avenue, Lakewood, N.J. 08701. Product Code
3048.) Ovalbumin peptide 323-339 amide acetate salt, was purchased
from Bachem Americas Inc. (3132 Kashiwa Street, Torrance Calif.
90505. Product code 4065609.) PLGA-R848 of approximately 5,200 Da
made from PLGA of 3:1 lactide to glycolide ratio and having 12.7%
w/w conjugated R848 content was synthesized. PLA-PEG-OMe block
co-polymer with a methyl ether terminated PEG block of 2,000 Da and
DL-PLA block of approximately 19,000 Da was synthesized. PLA with
an inherent viscosity of 0.21 dL/g was purchased from SurModics
Pharmaceuticals (756 Tom Martin Drive, Birmingham, Ala. 35211.
Product Code 100 DL 2A.) Polyvinyl alcohol (Mw=11,000-31,000,
87-89% hydrolyzed) was purchased from J. T. Baker (Part Number
U232-08).
Method
Solutions were prepared as follows:
Solution 1A: Ovalbumin protein at 40 mg/mL in 10 mM phosphate
buffer.
Solution 1B: Ovalbumin peptide amide 323-339 @ 40 mg/mL in dilute
hydrochloric acid aqueous solution.
Solution 2: PLGA-R848 at 50 mg/mL, PLA-PEG-OMe at 25 mg/mL, PLA at
25 mg/ml in dichloromethane. The solution was prepared by
separately dissolving each polymer as a 100 mg/mL in
dichloromethane, then mixing the solutions by adding 2 parts
PLGA-R848 solution to 1 part each PLA-PEG-OMe solution and PLA
solution.
Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM in 100 mM
phosphate buffer, pH 8.
Solution 4: 70 mM phosphate buffer, pH 8.
The first primary (W1/O) emulsion was created using Solution 1A
& Solution 2. Solution 1A (0.2 mL) and Solution 2 (1.0 mL) were
combined in a small glass pressure tube and sonicated at 50%
amplitude for 40 seconds using a Branson Digital Sonifier 250. A
second primary (W1/O) emulsion was created using Solution 1B &
Solution 2. Solution 1B (0.2 mL) and Solution 2 (1.0 mL) were
combined in a small glass pressure tube and sonicated at 50%
amplitude for 40 seconds using a Branson Digital Sonifier 250. The
two primary emulsions were combined, in part, by transferring 0.6
mL of each into a third glass pressure tube to create a 1:1 mixture
of the two primary emulsions in a total volume of approximately 1.2
mL. A secondary (W1/O/W2) emulsion was then formed by adding
Solution 3 (2.0 mL) to the primary emulsion, and then sonicating at
30% amplitude for 40 seconds using the Branson Digital Sonifier
250. The secondary emulsion was added to an open 50 mL beaker
containing 70 mM phosphate buffer solution (30 mL) and stirred at
room temperature for 2 hours to allow the dichloromethane to
evaporate and the nanocarriers to form in suspension. A portion of
the suspended nanocarriers was washed by transferring the
nanocarrier suspension to a centrifuge tube, spinning at 13,823 g
for one hour, removing the supernatant, and re-suspending the
pellet in phosphate buffered saline. This washing procedure was
repeated and then the pellet was re-suspended in phosphate buffered
saline to achieve a nanocarrier suspension having a nominal
concentration of 10 mg/mL on a polymer basis. The suspension was
stored frozen at -20.degree. C. until use.
TABLE-US-00004 TABLE 4 Nanocarrier Characterization Ovalbumin
Ovalbumin Nanocarrier Effective R848 Protein Peptide ID Diameter
(nm) (% w/w) (% w/w) (% w/w) 4 213 3.8 0.3 0.8
Example 5
Synthetic Nanocarrier Formulation Lot #5
Materials
SIINFEKL (SEQ ID NO: 1) (ovalbumin peptide [257-264]), was
purchased from Bachem Americas Inc. (3132 Kashiwa Street, Torrance
Calif. 90505. Product code H-4866.) Ovalbumin peptide 323-339 amide
acetate salt, was purchased from Bachem Americas Inc. (3132 Kashiwa
Street, Torrance Calif. 90505. Product code 4065609.) PLGA-R848 of
approximately 4,500 Da made from PLGA of 3:1 lactide to glycolide
ratio and having 15% w/w conjugated R848 content was synthesized.
PLA-PEG-Nicotine with a nicotine-terminated PEG block of
approximately 5,000 Da and DL-PLA block of approximately 17,000 Da
was synthesized. PLA with an inherent viscosity of 0.21 dL/g was
purchased from SurModics Pharmaceuticals (756 Tom Martin Drive,
Birmingham, Ala. 35211. Product Code 100 DL 2A.) Polyvinyl alcohol
(Mw=11,000-31,000, 87-89% hydrolyzed) was purchased from J. T.
Baker (Part Number U232-08).
Method
Solutions were prepared as follows:
Solution 1A: SIINFEKL (SEQ ID NO: 1) @ 200 mg/mL DMSO.
Solution 1B: Ovalbumin peptide amide 323-339 @ 20 mg/mL in dilute
hydrochloric acid aqueous solution.
Solution 2: PLGA-R848 at 50 mg/mL, PLA-PEG-Nicotine at 25 mg/mL,
PLA at 25 mg/ml in dichloromethane. The solution was prepared by
separately dissolving each polymer as a 100 mg/mL in
dichloromethane, then mixing the solutions by adding 2 parts
PLGA-R848 solution to 1 part each PLA-PEG-Nicotine solution and PLA
solution.
Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM in 100 mM
phosphate buffer, pH 8.
Solution 4: 70 mM phosphate buffer, pH 8.
The first primary (S/O) emulsion was created using Solution 1A
& Solution 2. Solution 1A (0.025 mL) and Solution 2 (1.0 mL)
were combined in a small glass pressure tube and sonicated at 50%
amplitude for 40 seconds using a Branson Digital Sonifier 250. A
serial primary (W1/(S/O)) emulsion was created using Solution 1B
and the first primary emulsion. Solution 1B (0.25 mL) was added to
the small glass pressure tube containing the first primary emulsion
and then sonicated at 50% amplitude for 40 seconds using a Branson
Digital Sonifier 250. A secondary ((S+W1)/O/W2) emulsion was then
formed by adding Solution 3 (2.0 mL) to the serial primary
emulsion, and then sonicating at 30% amplitude for seconds using
the Branson Digital Sonifier 250. The secondary emulsion was added
to an open 50 mL beaker containing 70 mM phosphate buffer solution
(30 mL) and stirred at room temperature for 2 hours to allow the
dichloromethane to evaporate and the nanocarriers to form in
suspension. A portion of the suspended nanocarriers was washed by
transferring the nanocarrier suspension to a centrifuge tube,
spinning at 13,823 g for one hour, removing the supernatant, and
re-suspending the pellet in phosphate buffered saline. This washing
procedure was repeated and then the pellet was re-suspended in
phosphate buffered saline to achieve a nanocarrier suspension
having a nominal concentration of 10 mg/mL on a polymer basis. The
suspension was stored frozen at -20.degree. C. until use.
TABLE-US-00005 TABLE 5 Nanocarrier Characterization Nano- Effective
SIINFEKL (SEQ Ovalbumin carrier Diameter R848 ID NO: 1) peptide
323-339 peptide ID (nm) (% w/w) (% w/w) (% w/w) 5 236 4.2 0.9
1.6
Example 6
Synthetic Nanocarrier Compositions Generate High Antibody Titers
and Strong Antigen-Specific CTL Activity
3.sup.rd and 4.sup.th in vivo (C57BL/6 mice) immunization studies
were performed. The above polymeric nanocarrier formulation (#3)
delivering a TLR agonist (R848) and entrapped ovalbumin protein
(OVA) was introduced and created high antibody titers (e.g.,
anti-OVA IgG titers of .about.1e6) and strong antigen-specific CTL
activity from local lymph and spleen cells.
Immunized mice were bled at dates indicated and antibodies to
ovalbumin measured in standard ELISA using serial dilutions of test
sera. Biotinylated goat anti-mouse Ig was used as a detection
antibody (BD Biosciences, San Diego, Calif.). EC50 was determined
based on titration curves. CTL activity was measured as follows.
4-5 days after the final injection (subcutaneous, s.c., or
intranasal, i.n.) with the nanocarrier preparations or protein
controls draining lymph nodes (LNs) were removed, treated with
collagenase, homogenized, washed and incubated with 10-100 units/ml
of IL-2 for 4-5 days. Then resulting cell populations were counted
and used as effector cells in cytotoxicity assays. Syngeneic EL-4
cells pulsed with SIINFEKL (SEQ ID NO: 1) peptide or EG.7-OVA cells
(stably transfected with ovalbumin) served as targets with intact
EL-4 cells providing for background control. Cytoxicity at various
effector:target ratios was measured over 24 hours (37.degree. C.)
using CytoTox-ONE.TM. Homogenuous Membrane Integrity Assay
(Promega, Madison, Wis.) according to manufacturer's
recommendations.
TABLE-US-00006 TABLE 6 Formulation of Nanocarrier Antigen OVA
Protein TLR Agonist PLGA-R848 (50%) Matrix polymer 1 PLA-PEG (25%)
Matrix Polymer 2 100 DL 2A (25%)
TABLE-US-00007 TABLE 7 Experiment 1 Layout Ova Ova NC R848 Protein
peptide Gr. # Immunized with Lot# load (%) load (%) load (%) 1 NC
(ovalbumin; no 1 4.0 1.1 N/A memory peptide) 2 NC (ovalbumin; 2 3.9
0.3 2.3 +memory peptide) 3 NC (ovalbumin; no 3 4.3 0.8 N/A memory
peptide) 4 NC (ovalbumin; 4 3.8 0.3 0.8 +memory peptide) 5
Ovalbumin (100 .mu.g) + N/A N/A 100 .mu.g N/A 20 .mu.g free CpG 6
Ovalbumin (100 .mu.g) + N/A N/A 100 .mu.g N/A 100 .mu.g alum
TABLE-US-00008 TABLE 8 Experiment 2 layout R848 Ova Ova peptide Gr.
NC load Protein load (%), # Immunized with Route Lot# (%) load (%)
type 1 NC (SIINFEKL S.c. 5 4.2 N/A 1.6-peptide (SEQ ID NO: 1) +
0.9- memory peptide) SIINFEKL (SEQ ID NO: 1) 2 NC (ovalbumin; S.c 3
4.3 0.8 N/A no memory peptide) 3 NC (SIINFEKL I.n. 5 4.2 N/A
1.6-peptide (SEQ ID NO: 1) + 0.9- memory peptide) SIINFEKL (SEQ ID
NO: 1) 4 NC (ovalbumin; I.n. 3 4.3 0.8 N/A no memory peptide)
Example 7
Synthetic Nanocarrier Formulation Lot #6
Materials
Ovalbumin protein, was purchased from Worthington Biochemical
Corporation (730 Vassar Avenue, Lakewood, N.J. 08701). Product Code
LS003054. PLGA-R848, Poly-D/L-lactide-co-glycolide,
4-amino-2-(ethoxymethyl)-.alpha.,.alpha.-dimethyl-1H-imidazo[4,5-c]quinol-
ine-1-ethanol amide of approximately 7,800 Da made from PLGA of 3:1
lactide to glycolide ratio and having 8.5% w/w conjugated
resiquimod content was custom manufactured at Princeton Global
Synthesis (300 George Patterson Drive #206, Bristol, Pa. 19007.)
Lot number PGS 16-52. PLA with an inherent viscosity of 0.21 dL/g
was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive,
Birmingham, Ala. 35211. Product Code 100 DL 2A.) PLA-PEG-OMe block
co-polymer with a methyl ether terminated PEG block of
approximately 5,000 Da and PLA block of approximately 21,000 Da by
.sup.1H-NMR (Mn of 21 kDa) was synthesized. EMPROVE.RTM. Polyvinyl
Alcohol 5-88, USP (85-89% hydrolyzed, viscosity of 4.3-5.7 mPas)
was purchased from EMD Chemicals Inc. (480 South Democrat Road
Gibbstown, N.J. 08027. Part Number 1.41354). Phosphate-buffered
saline 1.times. (PBS 1.times.). From Mediatech Inc. (9345 Discovery
Blvd. Manassas, Va. 20109.) Product Code 21-040-CV.
Method
Solutions were prepared as follows:
Solution 1: Ovalbumin protein @ 20 mg/mL was prepared in PBS
1.times. at room temperature.
Solution 2: PLA was prepared by dissolving PLA at 100 mg per 1 mL
of dichloromethane in the chemical fume hood.
Solution 3: PLA-PEG-OMe was prepared by dissolving PLA-PEG-OMe at
100 mg per 1 mL of dichloromethane in the chemical fume hood.
Solution 4: PLGA-R848 was prepared by dissolving PLGA-R848 at 100
mg per 1 mL of dichloromethane in the chemical fume hood.
Solution 5: Polyvinyl alcohol @ 50 mg/mL in 100 mM in 100 mM
phosphate buffer, pH 8.
Solution 6: 70 mM phosphate buffer, pH 8.
This lot was prepared in duplicate, then combined after washing. A
primary (W1/O) emulsion was first created by mixing Solutions 1
through 4. Solution 1 (0.2 mL), Solution 2 (0.50 mL), Solution 3
(0.25 mL) and Solution 4 (0.25 mL) were combined in a small glass
pressure tube and sonicated at 50% amplitude for 40 seconds using a
Branson Digital Sonifier 250. A secondary (W1/O/W2) emulsion was
then formed by adding Solution 5 (2.0 mL) to the primary emulsion,
vortexing to create a crude dispersion, and then sonicating at 30%
amplitude for 60 seconds using the Branson Digital Sonifier 250.
The secondary emulsion was added to an open 50 mL beaker containing
Solution 6 (30 mL) and stirred at room temperature for 2 hours to
allow the dichloromethane to evaporate and the nanocarriers to form
in suspension. A portion of the suspended nanocarriers was washed
by transferring the nanocarrier suspension to a centrifuge tube,
spinning at 21,000 rcf for 45 minutes, removing the supernatant,
and re-suspending the pellet in phosphate buffered saline. This
washing procedure was repeated and then the pellet was re-suspended
in PBS 1.times. to achieve a nanocarrier suspension having a
nominal concentration of 10 mg/mL on a polymer basis. The
suspension was stored frozen at -20 C until use.
TABLE-US-00009 TABLE 9 Nanocarrier Characterization Nanocarrier
Effective TLR Agonist, Antigen, ID Diameter (nm) % w/w % w/w 6
252.1 R848, 4.4 OVA protein, 4.3
Example 8
Synthetic Nanocarrier Formulation Lot #7
Materials
Ovalbumin protein, was purchased from Worthington Biochemical
Corporation (730 Vassar Avenue, Lakewood, N.J. 08701). Product Code
LS003054. PLGA-R848, Poly-D/L-lactide-co-glycolide,
4-amino-2-(ethoxymethyl)-.alpha.,.alpha.-dimethyl-1H-imidazo[4,5-c]quinol-
ine-1-ethanol amide of approximately 7,800 Da made from PLGA of 3:1
lactide to glycolide ratio and having 8.5% w/w conjugated
resiquimod content was custom manufactured at Princeton Global
Synthesis (300 George Patterson Drive #206, Bristol, Pa. 19007.)
Lot number PGS 16-52. PLA with an inherent viscosity of 0.21 dL/g
was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive,
Birmingham, Ala. 35211. Product Code 100 DL 2A.) PLA-PEG-OMe block
co-polymer with a methyl ether terminated PEG block of
approximately 5,000 Da and PLA block of approximately 21,000 Da by
.sup.1H-NMR (Mn of 21 kDa) was synthesized. EMPROVE.RTM. Polyvinyl
Alcohol 5-88, USP (85-89% hydrolyzed, viscosity of 4.3-5.7 mPas)
was purchased from EMD Chemicals Inc. (480 South Democrat Road
Gibbstown, N.J. 08027. Part Number 1.41354). Phosphate-buffered
saline 1.times. (PBS 1.times.). From Mediatech Inc. (9345 Discovery
Blvd. Manassas, Va. 20109.) Product Code 21-040-CV.
Method
Solutions were prepared as follows:
Solution 1: Ovalbumin protein @ 5 mg/mL was prepared in PBS
1.times. at room temperature.
Solution 2: PLA was prepared by dissolving PLA at 100 mg per 1 mL
of dichloromethane in the chemical fume hood.
Solution 3: PLA-PEG-OMe was prepared by dissolving PLA-PEG-OMe at
100 mg per 1 mL of dichloromethane in the chemical fume hood.
Solution 4: PLGA-R848 was prepared by dissolving PLGA-R848 at 100
mg per 1 mL of dichloromethane in the chemical fume hood.
Solution 5: Polyvinyl alcohol @ 50 mg/mL in 100 mM in 100 mM
phosphate buffer, pH 8.
Solution 6: 70 mM phosphate buffer, pH 8.
This lot was prepared in duplicate, then combined after washing. A
primary (W1/O) emulsion was first created by mixing Solutions 1
through 4. Solution 1 (0.2 mL), Solution 2 (0.50 mL), Solution 3
(0.25 mL) and Solution 4 (0.25 mL) were combined in a small glass
pressure tube and sonicated at 50% amplitude for 40 seconds using a
Branson Digital Sonifier 250. A secondary (W1/O/W2) emulsion was
then formed by adding Solution 5 (2.0 mL) to the primary emulsion,
vortexing to create a crude dispersion, and then sonicating at 30%
amplitude for 60 seconds using the Branson Digital Sonifier 250.
The secondary emulsion was added to an open 50 mL beaker containing
Solution 6 (30 mL) and stirred at room temperature for 2 hours to
allow the dichloromethane to evaporate and the nanocarriers to form
in suspension. A portion of the suspended nanocarriers was washed
by transferring the nanocarrier suspension to a centrifuge tube,
spinning at 21,000 rcf for 45 minutes, removing the supernatant,
and re-suspending the pellet in phosphate buffered saline. This
washing procedure was repeated and then the pellet was re-suspended
in PBS 1.times. to achieve a nanocarrier suspension having a
nominal concentration of 10 mg/mL on a polymer basis. The
suspension was stored frozen at -20 C until use.
TABLE-US-00010 TABLE 10 Nanocarrier Characterization Nanocarrier
Effective TLR Agonist, Antigen, ID Diameter (nm) % w/w % w/w 7
240.6 R848, 4.2 OVA protein, 1.3
Example 9
Synthetic Nanocarrier Formulation Lot #8
Materials
Ovalbumin protein, was purchased from Worthington Biochemical
Corporation (730 Vassar Avenue, Lakewood, N.J. 08701). Product Code
LS003054. PLGA-R848, Poly-D/L-lactide-co-glycolide,
4-amino-2-(ethoxymethyl)-.alpha.,.alpha.-dimethyl-1H-imidazo[4,5-c]quinol-
ine-1-ethanol amide of approximately 7,800 Da made from PLGA of 3:1
lactide to glycolide ratio and having 8.5% w/w conjugated
resiquimod content was custom manufactured at Princeton Global
Synthesis (300 George Patterson Drive #206, Bristol, Pa. 19007.)
PLA with an inherent viscosity of 0.21 dL/g was purchased from
SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, Ala.
35211. Product Code 100 DL 2A.) PLA-PEG-OMe block co-polymer with a
methyl ether terminated PEG block of approximately 5,000 Da and PLA
block of approximately 21,000 Da by .sup.1H-NMR (Mn of 21 kDa) was
synthesized. EMPROVE.RTM. Polyvinyl Alcohol 5-88, USP (85-89%
hydrolyzed, viscosity of 4.3-5.7 mPas) was purchased from EMD
Chemicals Inc. (480 South Democrat Road Gibbstown, N.J. 08027. Part
Number 1.41354). Phosphate-buffered saline 1.times. (PBS 1.times.).
From Mediatech Inc. (9345 Discovery Blvd. Manassas, Va. 20109.)
Product Code 21-040-CV.
Method
Solutions were prepared as follows:
Solution 1: Ovalbumin protein @ 20 mg/mL was prepared in PBS
1.times. at room temperature.
Solution 2: PLA was prepared by dissolving PLA at 100 mg per 1 mL
of dichloromethane in the chemical fume hood.
Solution 3: PLA-PEG-OMe was prepared by dissolving PLA-PEG-OMe at
100 mg per 1 mL of dichloromethane in the chemical fume hood.
Solution 4: PLGA-R848 was prepared by dissolving PLGA-R848 at 100
mg per 1 mL of dichloromethane in the chemical fume hood.
Solution 5: Polyvinyl alcohol @ 50 mg/mL in 100 mM in 100 mM
phosphate buffer, pH 8.
Solution 6: 70 mM phosphate buffer, pH 8.
This lot was prepared in duplicate, then combined after washing. A
primary (W1/O) emulsion was first created by mixing Solutions 1
through 4. Solution 1 (0.2 mL), Solution 2 (0.50 mL), Solution 3
(0.25 mL) and Solution 4 (0.25 mL) were combined in a small glass
pressure tube and sonicated at 50% amplitude for 40 seconds using a
Branson Digital Sonifier 250. A secondary (W1/O/W2) emulsion was
then formed by adding Solution 5 (2.0 mL) to the primary emulsion,
vortexing to create a crude dispersion, and then sonicating at 30%
amplitude for 60 seconds using the Branson Digital Sonifier 250.
The secondary emulsion was added to an open 50 mL beaker containing
Solution 6 (30 mL) and stirred at room temperature for 2 hours to
allow the dichloromethane to evaporate and the nanocarriers to form
in suspension. A portion of the suspended nanocarriers was washed
by transferring the nanocarrier suspension to a centrifuge tube,
spinning at 21,000 rcf for 45 minutes, removing the supernatant,
and re-suspending the pellet in phosphate buffered saline. This
washing procedure was repeated and then the pellet was re-suspended
in PBS 1.times. to achieve a nanocarrier suspension having a
nominal concentration of 10 mg/mL on a polymer basis. The
suspension was stored frozen at -20 C until use.
TABLE-US-00011 TABLE 11 Nanocarrier Characterization Nanocarrier
Effective TLR Agonist, Antigen, ID Diameter (nm) % w/w % w/w 8
238.6 R848, 3.9 OVA protein, 8.0
Example 10
Synthetic Nanocarrier Compositions Generate High Antibody Titers
and Strong Antigen-Specific CTL Activity
Synthetic nanocarriers delivering R848 and OVA was as successful as
a positive comparator control consisting of high dose of PS-CpG
plus a 6.times. higher dose of free OVA at generating a central
(spleen) OVA-specific CTL response and also in creating as strong
(or stronger) OVA-specific humoral response.
4-5 days after s.c. injection with nanocarrier preparations or
controls draining lymph nodes (LNs) were removed, treated with
collagenase, homogenized, washed and incubated with 10-100 units/ml
of IL-2 for 4-5 days. Then resulting cell populations were counted
and used as effector cells in cytotoxicity assay. Syngeneic EL-4
cells pulsed with SIINFEKL (SEQ ID NO: 1) peptide or EG.7-OVA cells
(stably transfected with ovalbumin) served as targets with intact
EL-4 cells providing for background control. Cytoxicity at various
effector:target ratios was measured over 24 hours (37.degree. C.)
using CytoTox-ONE.TM. Homogenuous Membrane Integrity Assay
(Promega, Madison, Wis.) according to manufacturer's
recommendations.
TABLE-US-00012 TABLE 12 Gr. # Immunized w. NC Lot Adjuvant (.mu.g)
OVA (.mu.g) 1 NC-OVA-R848 6 R848 (4.4) 4.3 2 NC-OVA-R848 7 R848
(4.2) 1.3 3 NC-OVA-R848 8 R848 (3.9) 8.0 4 OVA + CpG N/A CpG, 20
.mu.g 50
Example 11
Measure Development of Humoral and Cellular Immune Responses to
Nanocarrier-encapsulated Antigen After a Single Injection of
NC-OVA+NC-R848 Mix
Materials--Lot #9
PLGA-R848 (S-205), Poly-D/L-lactide-co-glycolide,
4-amino-2-(ethoxymethyl)-.alpha.,.alpha.-dimethyl-1H-imidazo[4,5-c]quinol-
ine-1-ethanol amide of approximately 7,800 Da made from PLGA of 3:1
lactide to glycolide ratio and having 8.5% w/w conjugated
resiquimod content was custom manufactured at Princeton Global
Synthesis (300 George Patterson Drive #206, Bristol, Pa. 19007.).
PLA with an inherent viscosity of 0.19 dL/g was purchased from
SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, Ala.
35211. Product Code 100 DL 2A). PLA-PEG-OMe block co-polymer with a
methyl ether terminated PEG block of approximately 5,000 Da and PLA
block of approximately 28,000 Da was purchased from SurModics
Pharmaceuticals (Product Code 100 DL mPEG 5000 5CE). EMPROVE.RTM.
Polyvinyl Alcohol 4-88, USP (85-89% hydrolyzed, viscosity of
3.4-4.6 mPas) was purchased from EMD Chemicals Inc. (480 South
Democrat Road Gibbstown, N.J. 08027). Phosphate-buffered saline
1.times. (PBS 1.times.). From Mediatech Inc. (9345 Discovery Blvd.
Manassas, Va. 20109.) Product Code 21-040-CV.
Method--Lot #9
Solutions were prepared as follows:
Solution 1: PLGA-R848 was prepared by weighing out PLGA-R848, PLA,
and PLA-PEG-OMe powders in 2:1:1 weight ratio and then dissolving
the mixed polymers in dichloromethane to achieve a total polymer
concentration of 100 mg per 1 mL.
Solution 2: Polyvinyl alcohol @ 35 mg/mL in 100 mM phosphate
buffer, pH 8.
An O/W emulsion was created by mixing Solutions 1 and 2, and then
creating a coarse emulsion prior to a fine emulsion. Solution 1 (2
mL) was coarsely emulsified with Solution 2 (8 mL), using 10 passes
through an 18 G emulsification needle. A fine emulsion was made by
loading the coarse emulsion into a primed and ice-water-chilled
high pressure homogenizer (Microfluidics LV1) and performing three
passes at 5000 psi. The fine O/W emulsion was added to an open 100
mL beaker containing 1.times.PBS (60 mL) and stirred at room
temperature for more than 2 hours to allow the dichloromethane to
evaporate and the nanocarriers to form in suspension. A portion of
the suspended nanocarriers was washed by transferring the
nanocarrier suspension to a centrifuge tube, spinning at 75,600 rcf
for 35 minutes, removing the supernatant, and re-suspending the
pellet in phosphate buffered saline. This washing procedure was
repeated and then the pellet was re-suspended in PBS 1.times. to
achieve a nanocarrier suspension having a nominal concentration of
10 mg/mL on a polymer basis. The nanocarrier formation process was
repeated another three times at 1.times. or 2.times. the same
scale. The four suspensions were combined and then filtered through
0.22 micron PES syringe filter and then stored frozen at -20 C
until use.
Materials--Lot #10
PLGA-R848 (S-205), Poly-D/L-lactide-co-glycolide,
4-amino-2-(ethoxymethyl)-.alpha.,.alpha.-dimethyl-1H-imidazo[4,5-c]quinol-
ine-1-ethanol amide of approximately 7,800 Da made from PLGA of 3:1
lactide to glycolide ratio and having 8.5% w/w conjugated
resiquimod content was custom manufactured at Princeton Global
Synthesis (300 George Patterson Drive #206, Bristol, Pa. 19007.).
PLA with an inherent viscosity of 0.19 dL/g was purchased from
SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, Ala.
35211. Product Code 100 DL 2A). PLA-PEG-OMe block co-polymer with a
methyl ether terminated PEG block of approximately 5,000 Da and PLA
block of approximately 28,000 Da was purchased from SurModics
Pharmaceuticals (Product Code 100 DL mPEG 5000 5CE). EMPROVE.RTM.
Polyvinyl Alcohol 4-88, USP (85-89% hydrolyzed, viscosity of
3.4-4.6 mPas) was purchased from EMD Chemicals Inc. (480 South
Democrat Road Gibbstown, N.J. 08027). Phosphate-buffered saline
1.times. (PBS 1.times.). From Mediatech Inc. (9345 Discovery Blvd.
Manassas, Va. 20109.) Product Code 21-040-CV.
Method--Lot #10
Solutions were prepared as follows:
Solution 1: PLGA-R848 was prepared by dissolving PLGA-R848 at 100
mg per 1 mL of dichloromethane in the chemical fume hood.
Solution 2: PLA was prepared by dissolving PLA at 100 mg per 1 mL
of dichloromethane in the chemical fume hood.
Solution 3: PLA-PEG-OMe was prepared by dissolving PLA-PEG-OMe at
100 mg per 1 mL of dichloromethane in the chemical fume hood.
Solution 4: Polyvinyl alcohol @ 50 mg/mL in 100 mM phosphate
buffer, pH 8.
A primary (O/W) emulsion was created by mixing Solutions 1 through
4 and creating a coarse emulsion prior to a fine emulsion. Solution
1 (1 mL), Solution 2 (0.5 mL), and Solution 3 (0.5 mL) were
combined first and then coarsely emulsified with Solution 4 (8 mL),
by stirring together at 350 rpm in a 50 mL beaker for two minutes
and by repeat pipetting. A fine emulsion, was made by loading the
coarse emulsion into a primed high pressure homogenizer
(Microfluidics LV1) and performing three passes at 5000 psi. The
fine O/W emulsion was added to an open 50 mL beaker containing
1.times.PBS (30 mL) and stirred at room temperature for more than 2
hours to allow the dichloromethane to evaporate and the
nanocarriers to form in suspension. A portion of the suspended
nanocarriers was washed by transferring the nanocarrier suspension
to a centrifuge tube, spinning at 75,600 rcf for 35 minutes,
removing the supernatant, and re-suspending the pellet in phosphate
buffered saline. This washing procedure was repeated and then the
pellet was re-suspended in PBS 1.times. to achieve a nanocarrier
suspension having a nominal concentration of 10 mg/mL on a polymer
basis. Nanocarrier suspension was then filtered through a 0.22
micron PES syringe filter and then stored frozen at -20 C until
use.
Materials--Lot #11
Ovalbumin protein, was purchased from Worthington Biochemical
Corporation (730 Vassar Avenue, Lakewood, N.J. 08701). Product Code
LS003054. PLGA with 75% lactide and 25% glycolide content and an
inherent viscosity of 0.24 dL/g was purchased from SurModics
Pharmaceuticals (756 Tom Martin Drive, Birmingham, Ala. 35211.
Product Code 7525 DLG 2.5A). PLA with an inherent viscosity of 0.2
dL/g was purchased from SurModics Pharmaceuticals (Product Code 100
DL 2A). PLA-PEG-OMe block co-polymer with a methyl ether terminated
PEG block of approximately 5,000 Da and PLA block of approximately
21,000 Da was synthesized. EMPROVE.RTM. Polyvinyl Alcohol 4-88, USP
(85-89% hydrolyzed, viscosity of 3.4-4.6 mPas) was purchased from
EMD Chemicals Inc. (480 South Democrat Road Gibbstown, N.J. 08027).
Phosphate-buffered saline IX (PBS IX). From Mediatech Inc. (9345
Discovery Blvd. Manassas, Va. 20109.) Product Code 21-040-CV.
Method--Lot #11
Solutions were prepared as follows:
Solution 1: Ovalbumin protein @ 50 mg/mL was prepared in PBS
1.times. at room temperature.
Solution 2: PLGA, PLA, and PLA-PEG-OMe were weighed out in 2:1:1
weight ratio and dissolved in dichloromethane in the chemical fume
hood to achieve a final total polymer concentration of 100 mg per 1
mL.
Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM phosphate
buffer, pH 8.
Solution 4: 70 mM phosphate buffer, pH 8.
A primary (W1/O) emulsion was first created by mixing Solutions 1
and 2. Solution 1 (66 mL) and Solution 2 (264 mL) were combined and
formed into a coarse emulsion using an overhead mixer in a 1 L
beaker. The coarse emulsion was transferred to a custom-made
temperature-controlled homogenization vessel and homogenized using
a high-shear rotor stator. A coarse secondary (W1/O/W2) emulsion
was then formed by transferring 2 of the primary emulsion into a
beaker containing 330 mL of Solution 3 and mixing with an overhead
mixer. The coarse secondary emulsion was then returned to a custom
homogenization vessel and homogenized to a fine emulsion using high
shear. The secondary emulsion was then added to a purged 6 L vessel
containing Solution 4 (3.2 L) and shaken overnight at room
temperature to evaporate and the nanocarriers to form in
suspension. Portions (30 mL each) of the suspended nanocarriers
were washed by transferring the nanocarrier suspension to a
centrifuge tube, spinning at 75,600 rcf for 35 minutes, removing
the supernatant, and re-suspending the pellet in phosphate buffered
saline. This washing procedure was repeated and then the pellet was
re-suspended in PBS 1.times. to achieve the target nanocarrier
concentration. The suspension was filtered and stored frozen at -20
C until use.
C57BL/6 female mice (2-3/group) were immunized once by a mix of 100
g NC-OVA and 100 g of NC-R848 containing 4.3 g R848 and 6.2 g of
OVA (per each mouse). At days 4, 7, 10 and 14 after nanocarrier
inoculation mice were bled and their antibody (IgG) titer against
OVA determined by ELISA. Additionally, at the same time-points mice
were injected (i.v.) by syngeneic splenocytes pulsed by a peptide
representing a dominant CTL epitope of OVA (SIINFEKL (SEQ ID NO:
1)) and differentially labeled by CSFE. The next day, splenocytes
from immunized mice were taken and analyzed by FACS and specific
cytotoxicity in each animal determined compared to basic
cytotoxicity level in PBS-injected (naive) animals
(%=100.times.[1-RRnaive/RRimm]).
A single immunization with NC-OVA+NC-R848 leads to rapid induction
of cellular and humoral immune responses with the former being
detected as early as four days after injection and then persisting
for at least ten days with a peak at day 7, and the latter being
detected at seven days after injection and reaching a significant
level at 10 days after inoculation.
Example 12
Synthetic Nanocarriers Delivering CpG and OVA
Materials--Lot #12
PO-1826 DNA oligonucleotide with phosphodiester backbone having
nucleotide sequence 5'-TCC ATG ACG TTC CTG ACG TT-3' (SEQ ID NO:2)
with a sodium counter-ion was purchased from Oligo Factory (120
Jeffrey Avenue, Holliston, Mass. 01746.) PLGA having 54% lactide
and 46% glycolide content and an inherent viscosity of 0.24 dL/g
was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive,
Birmingham, Ala. 35211. Product Code 5050 DLG 2.5A). PLGA-PEG-OMe
block co-polymer with a methyl ether terminated PEG block of
approximately 2,000 Da and 75% lactide/25% glycolide PLGA block of
approximately 88,000 Da was purchased from SurModics
Pharmaceuticals (Product Code 7525 DLG PEG 2000 7E-P). EMPROVE.RTM.
Polyvinyl Alcohol 4-88, USP (85-89% hydrolyzed, viscosity of
3.4-4.6 mPas) was purchased from EMD Chemicals Inc. (480 South
Democrat Road Gibbstown, N.J. 08027). Phosphate-buffered saline
1.times. (PBS 1.times.). From Mediatech Inc. (9345 Discovery Blvd.
Manassas, Va. 20109.) Product Code 21-040-CV.
Method--Lot #12
Solutions were prepared as follows:
Solution 1: PO-1826 was prepared by dissolving at 40 mg per 1 mL of
an aqueous solution containing 250 mg Na cholate per 1 mL
endotoxin-free water.
Solution 2: PLGA was prepared by dissolving PLA at 100 mg per 1 mL
of dichloromethane in the chemical fume hood.
Solution 3: PLGA-PEG-OMe was prepared by dissolving PLGA-PEG-OMe at
100 mg per 1 mL of dichloromethane in the chemical fume hood.
Solution 4: Polyvinyl alcohol @ 50 mg/mL in 100 mM phosphate
buffer, pH 8.
Solution 5: 70 mM phosphate buffer, pH 8.
A primary (W/O) emulsion was created by mixing Solutions 1 through
3 and creating a coarse emulsion prior to a fine emulsion. Solution
1 (0.2 mL), Solution 2 (0.5 mL), and Solution 3 (0.5 mL) were
combined in a small glass pressure tube, coarsely emulsified by
repeat pipetting, and sonicated at 50% amplitude for 40 seconds
over an ice bath using a Branson Digital Sonifier 250. A secondary
(W1/O/W2) emulsion was then formed by adding Solution 4 (3.0 mL) to
the primary emulsion, vortexing to create a crude dispersion, and
then sonicating at 30% amplitude for 60 seconds over an ice bath
using the Branson Digital Sonifier 250. The fine W1/O/W2 emulsion
was added to an open 50 mL beaker containing 70 mM phosphate buffer
(30 mL) and stirred at room temperature for 2 hours to allow the
dichloromethane to evaporate and the nanocarriers to form in
suspension. A portion of the suspended nanocarriers was washed by
transferring the nanocarrier suspension to a centrifuge tube,
spinning at 21,000 rcf for 90 minutes, removing the supernatant,
and re-suspending the pellet in phosphate buffered saline. This
washing procedure was repeated and then the pellet was re-suspended
in PBS 1.times. to achieve a nanocarrier suspension having a
nominal concentration of 10 mg/mL on a polymer basis. Nanocarrier
suspension was then filtered through 0.22 micron PES syringe
filters, stored refrigerated until the concentration was
determined, and then concentration adjusted and stored frozen at
-20 C until use.
Materials--Lot #13
Ovalbumin protein, was purchased from Worthington Biochemical
Corporation (730 Vassar Avenue, Lakewood, N.J. 08701). Product Code
LS003054. PLGA with 75% lactide and 25% glycolide content and an
inherent viscosity of 0.2 dL/g was purchased from SurModics
Pharmaceuticals (756 Tom Martin Drive, Birmingham, Ala. 35211.
Product Code 7525 DLG 2A). PLA-PEG-OMe block co-polymer with a
methyl ether terminated PEG block of approximately 5,000 Da and PLA
block of approximately 21,000 Da by .sup.1H-NMR (Mn of 21 kDa) was
synthesized. EMPROVE.RTM. Polyvinyl Alcohol 4-88, USP (85-89%
hydrolyzed, viscosity of 3.4-4.6 mPas) was purchased from EMD
Chemicals Inc. (480 South Democrat Road Gibbstown, N.J. 08027).
Phosphate-buffered saline IX (PBS IX). From Mediatech Inc. (9345
Discovery Blvd. Manassas, Va. 20109.) Product Code 21-040-CV.
Method--Lot #13
Solutions were prepared as follows:
Solution 1: Ovalbumin protein @ 50 mg/mL was prepared in PBS
1.times. at room temperature.
Solution 2: PLGA was prepared by dissolving PLGA at 100 mg per 1 mL
of dichloromethane in the chemical fume hood.
Solution 3: PLA-PEG-OMe was prepared by dissolving PLA-PEG-OMe at
100 mg per 1 mL of dichloromethane in the chemical fume hood.
Solution 4: Polyvinyl alcohol @ 50 mg/mL in 100 mM phosphate
buffer, pH 8.
Solution 5: 70 mM phosphate buffer, pH 8.
A primary (W1/O) emulsion was first created by mixing Solutions 1
through 3. Solution 1 (0.2 mL), Solution 2 (0.75 mL), and Solution
3 (0.25 mL) were combined in a small glass pressure tube and
sonicated at 50% amplitude for 40 seconds over an ice bath using a
Branson Digital Sonifier 250. A secondary (W1/O/W2) emulsion was
then formed by adding Solution 4 (3.0 mL) to the primary emulsion,
vortexing to create a crude dispersion, and then sonicating at 30%
amplitude for 60 seconds over an ice bath using the Branson Digital
Sonifier 250. The secondary emulsion was added to an open 50 mL
beaker containing Solution 5 (30 mL) and stirred at room
temperature for 2 hours to allow the dichloromethane to evaporate
and the nanocarriers to form in suspension. A portion of the
suspended nanocarriers was washed by transferring the nanocarrier
suspension to a centrifuge tube, spinning at 21,000 rcf for 130
minutes, removing the supernatant, and re-suspending the pellet in
phosphate buffered saline. This washing procedure was repeated and
then the pellet was re-suspended in PBS 1.times. to achieve a
nanocarrier suspension having a nominal concentration of 10 mg/mL
on a polymer basis. The suspension was stored frozen at -20 C until
use.
Materials--Lot #14
Ovalbumin protein, was purchased from Worthington Biochemical
Corporation (730 Vassar Avenue, Lakewood, N.J. 08701. Product Code
LS003054). PLGA with 76% lactide and 24% glycolide content and an
inherent viscosity of 0.69 dL/g was purchased from SurModics
Pharmaceuticals (756 Tom Martin Drive, Birmingham, Ala. 35211.
Product Code 7525 DLG 7A). PLA with an inherent viscosity of 0.22
dL/g was purchased from SurModics Pharmaceuticals (Product Code 100
DL 2A). PLA-PEG-OMe block co-polymer with a methyl ether terminated
PEG block of approximately 5,000 Da and PLA block of approximately
21,000 Da was synthesized. EMPROVE.RTM. Polyvinyl Alcohol 4-88, USP
(85-89% hydrolyzed, viscosity of 3.4-4.6 mPas) was purchased from
EMD Chemicals Inc. (480 South Democrat Road Gibbstown, N.J. 08027).
Phosphate-buffered saline 1.times. (PBS 1.times.) was purchased
from Mediatech Inc. (9345 Discovery Blvd. Manassas, Va. 20109.)
Product Code 21-040-CV.
Method--Lot #14
Solutions were prepared as follows:
Solution 1: Ovalbumin protein @ 50 mg/mL was prepared in PBS
1.times. at room temperature.
Solution 2: PLGA was prepared by dissolving PLGA at 100 mg per 1 mL
of dichloromethane in the chemical fume hood.
Solution 3: PLA was prepared by dissolving PLGA at 100 mg per 1 mL
of dichloromethane in the chemical fume hood.
Solution 4: PLA-PEG-OMe was prepared by dissolving PLA-PEG-OMe at
100 mg per 1 mL of dichloromethane in the chemical fume hood.
Solution 5: Polyvinyl alcohol @ 50 mg/mL in 100 mM phosphate
buffer, pH 8.
Solution 6: 70 mM phosphate buffer, pH 8.
A primary (W1/O) emulsion was first created by mixing Solutions 1
through 4. Solution 1 (0.2 mL), Solution 2 (0.5 mL), Solution 3
(0.25 mL) and Solution 4 (0.25 mL) were combined in a small glass
pressure tube and sonicated at 50% amplitude for 40 seconds over an
ice bath using a Branson Digital Sonifier 250. A secondary
(W1/O/W2) emulsion was then formed by adding Solution 5 (3.0 mL) to
the primary emulsion, vortexing to create a crude dispersion, and
then sonicating at 30% amplitude for 60 seconds over an ice bath
using the Branson Digital Sonifier 250. The secondary emulsion was
added to an open 50 mL beaker containing Solution 6 (30 mL) and
stirred at room temperature for 2 hours to allow the
dichloromethane to evaporate and the nanocarriers to form in
suspension. A portion of the suspended nanocarriers was washed by
transferring the nanocarrier suspension to a centrifuge tube,
spinning at 25,600 rcf for 45 minutes, removing the supernatant,
and re-suspending the pellet in phosphate buffered saline. This
washing procedure was repeated and then the pellet was re-suspended
in PBS 1.times. to achieve a nanocarrier suspension having a
nominal concentration of 10 mg/mL on a polymer basis. The
suspension was stored frozen at -20 C until use.
Materials--Lot #15
PO-1826 DNA oligonucleotide with phosphodiester backbone having
nucleotide sequence 5'-TCC ATG ACG TTC CTG ACG TT-3' (SEQ ID NO:2)
with a sodium counter-ion was purchased from Oligo Factory (120
Jeffrey Avenue, Holliston, Mass. 01746.) PLGA having 54% lactide
and 46% glycolide content and an inherent viscosity of 0.24 dL/g
was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive,
Birmingham, Ala. 35211. Product Code 5050 DLG 2.5A). PLA-PEG-OMe
block co-polymer with a methyl ether terminated PEG block of
approximately 5,000 Da and PLA block of approximately 21,000 Da was
synthesized. EMPROVE.RTM. Polyvinyl Alcohol 4-88, USP (85-89%
hydrolyzed, viscosity of 3.4-4.6 mPas) was purchased from EMD
Chemicals Inc. (480 South Democrat Road Gibbstown, N.J. 08027). Na
cholate was purchased from Sigma Aldrich LLC. (3050 Spruce St. St.
Louis, Mo. 6310. Product Code C6445-100G.) Phosphate-buffered
saline 1.times. (PBS 1.times.) was purchased from Mediatech Inc.
(9345 Discovery Blvd. Manassas, Va. 20109. Product Code
21-040-CV.)
Method--Lot #15
Solutions were prepared as follows:
Solution 1: PO-1826 was prepared by dissolving at 40 mg per 1 mL of
an aqueous solution containing 150 mg KCl per 1 mL of
endotoxin-free water.
Solution 2: Na Cholate was prepared by dissolving dry powder at 200
mg per 1 mL 1.times.PBS at room temperature.
Solution 3: PLGA was prepared by dissolving PLGA at 100 mg per 1 mL
of dichloromethane in the chemical fume hood.
Solution 4: PLGA-PEG-OMe was prepared by dissolving PLGA-PEG-OMe at
100 mg per 1 mL of dichloromethane in the chemical fume hood.
Solution 5: Polyvinyl alcohol @ 50 mg/mL in 100 mM phosphate
buffer, pH 8.
Solution 6: 70 mM phosphate buffer, pH 8.
A primary (W/O) emulsion was created by mixing Solutions 1 through
4 and creating a coarse emulsion prior to a fine emulsion. Solution
1 (0.25 mL), Solution 2 (0.25 mL), Solution 3 (0.5 mL), and
Solution 4 (0.5 mL) were combined in a small glass pressure tube,
coarsely emulsified by repeat pipetting, and sonicated at 50%
amplitude for 40 seconds over an ice bath using a Branson Digital
Sonifier 250. A secondary (W1/O/W2) emulsion was then formed by
adding Solution 5 (3.0 mL) to the primary emulsion, vortexing to
create a coarse dispersion, and then sonicating at 30% amplitude
for 60 seconds over an ice bath using the Branson Digital Sonifier
250. The fine W1/O/W2 emulsion was added to an open 50 mL beaker
containing 70 mM phosphate buffer (30 mL) and stirred at room
temperature for 2 hours to allow the dichloromethane to evaporate
and the nanocarriers to form in suspension. A portion of the
suspended nanocarriers was washed by transferring the nanocarrier
suspension to a centrifuge tube, spinning at 21,000 rcf for 90
minutes, removing the supernatant, and re-suspending the pellet in
phosphate buffered saline. This washing procedure was repeated and
then the pellet was re-suspended in PBS 1.times. to achieve a
nanocarrier suspension having a nominal concentration of 10 mg/mL
on a polymer basis. Nanocarrier suspension was then stored
refrigerated until the concentration was determined, and then
concentration adjusted and stored frozen at -20 C until use.
Materials--Lot #16
PO-1826 DNA oligonucleotide with phosphodiester backbone having
nucleotide sequence 5'-TCC ATG ACG TTC CTG ACG TT-3' (SEQ ID NO:2)
with a sodium counter-ion was purchased from Oligo Factory (120
Jeffrey Avenue, Holliston, Mass. 01746.) PLGA having 54% lactide
and 46% glycolide content and an inherent viscosity of 0.24 dL/g
was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive,
Birmingham, Ala. 35211. Product Code 5050 DLG 2.5A). PLA-PEG-OMe
block co-polymer with a methyl ether terminated PEG block of
approximately 5,000 Da and PLA block of approximately 21,000 Da was
synthesized. EMPROVE.RTM. Polyvinyl Alcohol 4-88, USP (85-89%
hydrolyzed, viscosity of 3.4-4.6 mPas) was purchased from EMD
Chemicals Inc. (480 South Democrat Road Gibbstown, N.J. 08027).
Phosphate-buffered saline 1.times. (PBS 1.times.) was purchased
from Mediatech Inc. (9345 Discovery Blvd. Manassas, Va. 20109.
Product Code 21-040-CV.)
Method--Lot #16
Solutions were prepared as follows:
Solution 1: PO-1826 was prepared by dissolving at 40 mg per 1 mL of
an aqueous solution containing 150 mg KCl per 1 mL of
endotoxin-free water.
Solution 2: Polyvinyl alcohol @ 100 mg/mL in 100 mM phosphate
buffer, pH 8.
Solution 3: PLGA was prepared by dissolving PLGA at 100 mg per 1 mL
of dichloromethane in the chemical fume hood.
Solution 4: PLGA-PEG-OMe was prepared by dissolving PLGA-PEG-OMe at
100 mg per 1 mL of dichloromethane in the chemical fume hood.
Solution 5: Polyvinyl alcohol @ 100 mg/mL in 100 mM phosphate
buffer, pH 8.
Solution 6: 70 mM phosphate buffer, pH 8.
A primary (W/O) emulsion was created by mixing Solutions 1 through
4 and creating a coarse emulsion prior to a fine emulsion. Solution
1 (0.25 mL), Solution 2 (0.25 mL), Solution 3 (0.5 mL), and
Solution 4 (0.5 mL) were combined in a small glass pressure tube,
coarsely emulsified by repeat pipetting, and sonicated at 50%
amplitude for 40 seconds over an ice bath using a Branson Digital
Sonifier 250. A secondary (W1/O/W2) emulsion was then formed by
adding Solution 5 (3.0 mL) to the primary emulsion, vortexing to
create a coarse dispersion, and then sonicating at 30% amplitude
for 60 seconds over an ice bath using the Branson Digital Sonifier
250. The fine W1/O/W2 emulsion was added to an open 50 mL beaker
containing 70 mM phosphate buffer (30 mL) and stirred at room
temperature for 2 hours to allow the dichloromethane to evaporate
and the nanocarriers to form in suspension. A portion of the
suspended nanocarriers was washed by transferring the nanocarrier
suspension to a centrifuge tube, spinning at 21,000 rcf for 90
minutes, removing the supernatant, and re-suspending the pellet in
phosphate buffered saline. This washing procedure was repeated and
then the pellet was re-suspended in PBS 1.times. to achieve a
nanocarrier suspension having a nominal concentration of 10 mg/mL
on a polymer basis. Nanocarrier suspension was then stored
refrigerated until the concentration was determined, and then
concentration adjusted and stored frozen at -20 C until use.
Synthetic nanocarriers delivering CpG and OVA were superior in
rapid antibody induction capacity than free high-dose CpG and OVA
(both free CpG and free OVA used in 5.times. higher dose) and as
successful or superior in induction of local and systemic
antigen-specific CTLs as the same high dose of free CpG and free
OVA.
Groups of three animals (C57BL/6 mice, females) were immunized
(prime-boost, days 0 and 10; hind limb, s.c.) by three combinations
of nanocarrier-incorporated CpG (ODN 1826) and OVA in parallel with
immunization with 5-fold excess of free CpG and OVA. Three
different formulations of NC-CpG were used with the same
formulation of NC-OVA (see Table 13 for details). At 4 days after
the second immunization animals were sacrificed and their serum,
draining (popliteal) lymph nodes (LNs) and spleens were taken. Sera
from immunized mice were used to determine antibody titer against
ovalbumin, which was measured in standard ELISA with serial
dilutions of test sera. Biotinylated goat anti-mouse Ig was used as
a detection antibody (BD Biosciences, San Diego, Calif.). EC50 was
determined based on titration curves. All NC-CpG formulations
combined with NC-OVA induced more than 30-fold higher early
antibody response against OVA than 5-fold higher doses of free CpG
and OVA.
In parallel, the induction of OVA-specific CTLs was assessed ex
vivo (without in vitro expansion) in draining LNs (locally) or
spleens (systemically) via FACS analysis. Briefly, both tissues
were treated with collagenase, homogenized, washed, cells counted
using Trypan exclusion (Countess, Invitrogen, CA, USA) and labeled
with antibodies or MHC class I-restricted pentamers coupled with
fluorescent dyes capable of recognizing surface CD8 (T cell
marker), CD19 (B cell marker) and T cell receptor (TCR) specific to
MHC Class-1-restricted immunodominant OVA-derived peptide SIINFEKL
(SEQ ID NO: 1).
Then differential cell populations were analyzed by FACS with those
cells exhibiting CD8 expression (CD8.sup.+), no CD19 expression
(CD19) and bound to MHC Class I-complexed SIINFEKL (SEQ ID NO: 1)
(SIINFEKL.sup.+ (SEQ ID NO: 1)) considered representing a major
species of OVA-specific CTLs. At least one of NC-CpG formulations
used has equal or higher capacity to produce short-term CTLs
locally and systemically when coupled with NC-OVA than 5-fold
higher amounts of free CpG and OVA. Of note, different NC-CpG
formulations have been especially beneficial for induction either
of local or of systemic CTL response against
nanocarrier-incorporated ovalbumin antigen.
Furthermore, purified splenocytes from immunized animals were also
expanded in vitro (100 u/mL IL-2) by being stimulated with
mitomycin-treated EG.7-OVA cells (syngeneic cells stably
transfected with ovalbumin), which should result in preferential
expansion of OVA-specific CD8.sup.+ cells. At 11 days of in vitro
incubation expanded cultures were labeled as described above and
analyzed by FACS. All NC-CpG formulations tested resulted in
induction of Ag-specific CTLs with a higher expansion potential
than those induced by 5.times. doses of free CpG and OVA. Of note,
one CpG formulation (NC-CpG) had especially strong potential for
CTL expansion and another (NC-CpG) has exceeded systemic CTL
induction levels by free high-dose CpG and OVA both when analyzed
ex vivo and upon in vitro expansion.
TABLE-US-00013 TABLE 13 Experimental layout for testing of humoral
and cellular immune response induced by nanocarrier- incorporated
CpG and OVA vs. free CpG and OVA. CpG OVA Gr. # Immunized w. NC Lot
(.mu.g) (.mu.g) Regimen 1 NC-OVA + NC-CpG #12/#13 4.0 10 0/10 d (1)
2 NC-OVA + NC-CpG #14/#15 4.0 10 Same (2) 3 NC-OVA + NC-CpG #14/#16
4.0 10 Same (3) 4 OVA + CpG (free) N/A 20 50 Same
SEQUENCE LISTINGS
1
218PRTArtificial SequenceSynthetic Peptide 1Ser Ile Ile Asn Phe Glu
Lys Leu1 5220PRTArtificial SequenceSynthetic Peptide 2Thr Cys Cys
Ala Thr Gly Ala Cys Gly Thr Thr Cys Cys Thr Gly Ala1 5 10 15Cys Gly
Thr Thr 20
* * * * *
References